UNASSIGNED

Upon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane.

UNASSIGNED

To evaluate procoagulant PS-exposing platelets by imaging flow cytometry.

UNASSIGNED

Platelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets were fluorescently labeled for the markers glycoprotein (GP)IX, activated integrin αIIbβ3, CD62P, and PS exposure.

UNASSIGNED

A subpopulation of platelets stimulated in suspension by the physiological agonists thrombin+collagen, and all platelets stimulated by the calcium ionophore A23187, had a distinct round morphology. These platelets were PS-exposing, larger in size, had an increased circularity index, and had reduced internal complexity compared with non-PS-exposing platelets. They expressed CD62P and αIIbβ3 in an inactive conformation on the surface, and demonstrated depolarized inner mitochondrial membranes. For the first time, using imaging flow cytometry, a large proportion of PS-exposing platelets possessing platelet-associated extracellular vesicles (EVs) was observed, which demonstrated heterogeneous platelet marker expression that was different from free released EVs.

UNASSIGNED

Innovative imaging flow cytometry allowed detailed fluorescence-based, quantitative morphometric analysis of PS-exposing platelets; in becoming procoagulant, platelets undergo remarkable morphological changes, transforming into spherical "balloons," almost devoid of their normal internal architecture. Almost all PS-exposing platelets have associated EVs that are not detectable by traditional flow cytometry. While their functions have yet to be fully elucidated, the heterogeneity of platelet-associated and released EVs suggests that they may contribute to different aspects of hemostasis and of thrombosis.

BACKGROUND

Based on the notion that full activation of platelets is required for a growth factor release, in regenerative dentistry, platelet-rich plasma (PRP) in liquid form is usually clotted by addition of CaCl2 in glassware before topical implantation. However, there has been no evidence as to which is better, full or partial activation of platelets, for minimizing the loss of growth factors and improving the controlled release of growth factors from coagulated PRP. To address this matter, here, we primarily examined direct effects of CaCl2 on platelets in PBS and on coagulation in citrated PRP.

METHODS

PRP was prepared from healthy volunteers' blood. Platelets' actions were monitored by scanning electron microscopy, flow cytometry, digital holographic microscopy, and immunofluorescent staining. Clot formation was examined in plasma.

RESULTS

In plasma-free PBS, 0.1% CaCl2 immediately upregulated CD62P and CD63, causing a release of microparticles and fibrinogen/fibrin; consequently, platelets aggregated and adhered to polystyrene culture dishes with enlargement of their attachment area. In a clot formation assay in plasma, CaCl2 initially induced platelet aggregation, which triggered loop-like matrix formation and subsequently induced coagulation on a watch glass. Such changes were not clearly observed either with PRP in a plastic dish or in platelet-poor plasma on a watch glass: coagulation was delayed in both conditions.

CONCLUSIONS

These findings indicate that besides the well-known coagulation pathway, which activates platelets via thrombin conversion in a coagulation cascade, CaCl2 directly activates platelets, which then facilitate clot formation independently and in cooperation with the coagulation pathway.

Refrigeration of platelets (4°C) provides the possibility of improving transfusion practice over the current standard-of-care, room temperature (RT) storage. However, the increased level of platelet activation observed at 4°C in vitro is cause for concern of uncontrolled thrombosis in vivo. In this study, we assessed the safety of 4°C-stored platelets by evaluating their response to physiologic inhibitors prostacyclin (PGI2) and nitric oxide (NO). Apheresis platelets were collected from healthy donors (n = 4) and tested on Day 1 (fresh) or Day 5 (RT- and 4°C-stored) after treatment with PGI2 and NO or not for: thrombin generation; factor V (FV) activity; intracellular free calcium, cAMP and cGMP; ATP release; TRAP-induced activation; aggregation to ADP, collagen, and TRAP, and adhesion to collagen under arterial flow. Data were analyzed using two-way ANOVA and post-hoc Tukey test for multiple comparisons, with significance set at P < 0.05. Treatment with inhibitors increased intracellular cAMP and cGMP levels in fresh and stored platelets. Thrombin generation was significantly accelerated in stored platelets consistent with increased factor V levels, PS exposure, CD62P expression, intracellular free calcium, and ATP release. While treatment with inhibitors did not attenuate thrombin generation in stored platelets, activation, aggregation, and adhesion responses were inhibited by both PGI2 and NO in 4°C-stored platelets. In contrast, though RT-stored platelets were activated, they did not adhere or aggregate in response to agonists. Thus, refrigerated platelets maintain their intracellular machinery, are responsive to agonists and platelet function inhibitors, and perform hemostatically better than RT-stored platelets.

BACKGROUND

We have studied the relationship between the ratio of activated platelets and the thickness of intima and media of the carotid artery in ischemic CVD patients in the chronic stage.

METHODS

Platelet activation was assessed by means of flow cytometry of whole blood using activation-dependent monoclonal antibodies (MoAb). Forty-one MRI-proven normative subjects and 55 patients with a history of ischemic CVD were examined. The intima-media thickness of the carotid artery was measured by using B-mode ultrasound in all subjects.

RESULTS

The appearance rates of PAC-1-positive and CD62P-positive platelets (%) were increased in ischemic CVD patients compared with those in controls (p<0.0001, p<0.001, respectively) The patients and controls were divided into those with atherosclerosis (Ath+), defined as intima-media thickness 1.1 mm, and those without (Ath-). There was no significant difference of PAC-1-positive platelets between the Ath- and Ath+ subgroups in either group, but there was increase in Ath- ischemic CVD patients versus Ath- control subjects (p<0.01), and in Ath+ patients versus Ath+ controls (p<0.05). CD62-positive platelets in the Ath+ subgroup were significantly increased versus the Ath- subgroup in both the controls (p<0.001) and ischemic CVD patients (p<0.05), and there was also an increase in Ath- patients versus Ath- controls (p<0.05).

CONCLUSIONS

Platelet activation markers were increased in patients with ischemic CVD compared with controls. A significant relationship was found between increased CD62-P-positive platelets and carotid artery abnormalities in both controls and ischemic CVD patients, suggesting that platelet activation may be a potential marker for atherosclerosis.

Increased serum concentrations of soluble intercellular adhesion molecule-1 (sICAM-1, CD54) and of soluble E- (CD62E), but not soluble P- (CD62P) and L- (CD62 L) selectins, were detected in Malagasy patients living in an hyperendemic focus of Schistosoma mansoni. Levels of sICAM-1 remained elevated for several months after treatment with praziquantel. Serum levels of ICAM-1, but not of other markers, were significantly correlated with the disease severity, as indicated by ultrasonographical data, and with some circulating fibrosis markers (at least hyaluronic acid). sICAM-1 level may reflect endothelial inflammatory reactions, probably harmful, in the liver and may be useful for monitoring morbidity evolution in schistosomiasis mansoni.

Cardiopulmonary bypass (CPB) elicits a systemic inflammatory response. The cause may include surface-induced leukocyte activation and hemolysis. A study was designed to describe the effects of both suction and an air-blood interface independently and in combination on leukocyte and platelet activation, and hemolysis in an in vitro model. Fresh human blood was drawn and tested in four different conditions including control (A), 10 minutes of -600 mm Hg suction (B), 10 minutes of blood exposure to room air at 100 ml/min (C), and 10 minutes of simultaneous suction and air flow (D). Samples were analyzed by flow cytometry (platelets and leukocytes) and plasma-free hemoglobin (PFHb). Leukocyte CD11b expression and platelet P-selectin (CD62P) were analyzed by flow cytometry. In comparison with baseline, granulocytes were significantly activated by air (group C, p = 0.0029) and combination (group D, p = 0.0123) but not by suction alone (group B). Monocytes and platelets were not significantly activated in any group. The PFHb increased significantly in group C (p < 0.001) and group D (p < 0.001). This study suggests that the inflammatory response and associated hemolysis during CPB may be related to air exposure, which could be reduced by minimizing the air exposure of air to blood during cardiotomy suction.

Treatment with thalidomide is associated with vascular thrombosis. The effect of thalidomide on platelet activation is unclear, although the use of aspirin is justified for thromboprophylaxis. A study on platelet activation markers was done among multiple myeloma patients receiving thalidomide therapy with warfarin as thromboprophylaxis. Strict criteria and procedure were set to avoid misinterpretation of platelet activation other than due to the thalidomide's effect. Blood specimen pre and post thalidomide therapy were used for flow cytometric analysis. Platelet surface P-selectin, CD62P expression and PAC-1 (antibody that recognizes conformational change of the GPIIb/IIIa complex) were examined by using three-colour flowcytometer. Increased expression marker for PAC-1 was observed after 4 weeks of thalidomide treatment (P < 0.05) indicating one aspect of platelet activation activity seen in these patients. The mechanism of thrombosis by thalidomide is probably multifactorial and one of them is likely through platelet activation. Further study on the affected pathway/s in the platelet activation process would confirm the exact mechanism of thalidomide-induced thrombosis and potential extended usage of this drug in future.

Graft occlusion following peripheral vascular surgery is attributable to some combination of acute thrombosis, and progression of atherosclerosis: interactions between leukocytes and activated platelets may play a role in both of these processes. This investigation measured perioperative leukocyte-platelet conjugate formation, and leukocyte and platelet activation in 46 patients undergoing surgery for lower extremity peripheral vascular disease (PVD). All patients were followed for graft patency over the next 6 months; 27 patients had grafts that remained patent while 19 had graft occlusion. On postoperative day #1 (POD#1), the graft occlusion group demonstrated a significantly greater increase in circulating levels of both monocyteplatelet and neutrophil (PMN)-platelet conjugates compared to the patent graft patients (p=0.015 and 0.018, respectively). PMN activation, assessed by increases in surface CD11b expression, was also significantly increased on POD#1 in the graft occlusion group compared to the patent group (p=0.026). The percentage of circulating activated (CD62P+) platelets did not differ between groups, but patients with graft occlusion demonstrated a higher percentage of younger, reticulated plate-lets throughout the study period (p=0.008), indicating increased platelet turnover. We conclude that in the early postoperative period, leukocyte-platelet adhesion, PMN activation, and platelet turnover are significantly greater in PVD patients who go on to develop later graft occlusion. Cellular activation and heterotypic cell interactions in peripheral vascular surgery patients may be important in the etiologies of thrombosis and/or accelerated atherosclerosis leading to graft loss.

A suitable model for the preclinical study of human platelet production in vivo has not been available. NOD/SCID mice were characterized as representing an efficient engraftment model for human hematopoietic stem cells, which resulted in the production of human platelets. Here, we evaluated in vivo human thrombopoiesis and ex vivo human platelet functions in NOD/SCID mice transplanted with human cord blood (CB) CD34(+) cells. Human platelets and human CD45(+) cells appeared in peripheral blood of NOD/SCID mice from 4 wk after transplantation. Human platelets produced in these mice showed CD62P expression and the activation of GPIIb/IIIa on human platelets on stimulation with an agonist. PEG-rHuMGDF (0, 0.5 and 5 microg/kg/d s.c.) was injected for 14 d into mice that had been confirmed to produce human platelets stably. The number of human platelets increased about twofold at 0.5 microg/kg/d and about fivefold at 5 microg/kg/d after 14 d. Withdrawal of PEG-rHuMGDF administration caused the human platelet count to return to the pretreatment level. Further, re-administration of PEG-rHuMGDF induced a similar human thrombopoietic response as it did on initial administration. These results suggest that NOD/SCID mice engrafted with human CB CD34(+) cells will be useful for the study of human platelet production in vivo.

An unsolved problem when employing small-diameter vascular grafts for aorto-coronary by-pass and peripheral reconstruction is the early thrombotic occlusion. The PEtU-PDMS is a new elastomeric material, composed of poly(ether)urethane and polydimethylsiloxane, synthesized to realize grafts with improved hemocompatibility characteristics. In order to investigate the effect of PDMS content on hemocompatibility, three different percentages of PDMS containing grafts (10, 25 and 40) were evaluated. Grafts realized with Estane 5714-F1 and silicone medical grade tubes were used as references. The hemocompatibility was investigated by an in vitro circuit in which human anticoagulated blood was circulated into grafts by a peristaltic pump modified to obtain a passive flow. For each experiment, 40 cm length graft was closed into a circular loop and put in rotation for 2 h at 37 degrees C. At the end of the experiments different parameters regarding platelet adhesion and activation were evaluated: circulating platelets count, beta-thromboglobulin release, platelet CD62P expression and amount of monocyte-platelet conjugates. PEtU-PDMS grafts with 25 and 40% of PDMS induced the lowest platelet adhesion, plasma level of beta-TG and amount of monocyte-platelet conjugates. No significative variations were observed in CD62P expression. In conclusion, PDMS content significatively affects blood-graft surface interaction, in fact higher PDMS percentage containing grafts showed the best in vitro hemocompatibility.

OBJECTIVE

Cardiac pump function and coronary regulation can be impaired after short-term ischemia. Recent studies with platelets (P) and neutrophils (PMN) yielded contradicting results about the "cellular" contribution to reperfusion injury.

METHODS

Isolated guinea pig hearts performing pressure-volume work were employed, external heart work (EHW), aortic flow (AF), coronary flow (CF) and heart rate (HR) serving as parameters of cardiac function. After global ischemia, human blood cells were given as bolus (1 min) during reperfusion (intracoronary hematocrit 7%). Expression of specific adhesion molecules (P: CD62P, CD41; PMN: integrin CD11b) was measured on cells before and after coronary passage (FACS analysis).

RESULTS

Postischemic recovery of pump function was significantly reduced in hearts with blood cell application (EHW: -cells 54 +/- 14%, +cells 41 +/-12%, p <0.05). Coronary response to bradykinin and reactive hyperemia were not effected. The blood-cell dependent functional loss was partly reduced by blocking CD18 (anti-CD 18) and completely abrogated by blockage of CD41 (lamifiban). The expression of CD11b on PMN and monocytes (M) and CD62P on platelets was significantly reduced in the coronary effluent and a significant decrease of CD41 on leukocytes occurred during coronary passage after ischemia. Increases in CD41 on PMN in the presence of lamifiban demasked intracoronary formation of micro aggregates (P/PMN). These micro aggregates were visualized by light microscopy. Electron microscopy revealed no significant microvascular plugging.

CONCLUSIONS

1) A specifically blood-cell induced loss of myocardial pump function has been demonstrated after short-term ischemia. 2) CD41 (= GpIIbIIIa) on P is responsible for this cardiac reperfusion damage. 3) The effect is causally linked to the formation of micro aggregates between PMN and P, but seems attenuated in the presence of erythrocytes as compared to effects reported from experiments in which PMN and P were applied singly or co-perfused. 4) Intracoronary retention of PMN, M and platelet-leukocyte micro aggregates seems to be transient, as adherence was not confirmed by electron microscopy.

BACKGROUND

Inflammation plays a key role in atherosclerotic disease. Up until now only limited evidence exists on the mechanism of cardiovascular complications in patients with depression. In addition depression was also linked to an increase in cardiovascular mortality. The present study was designed to evaluate the extent of platelet activation and platelet-derived markers of atherosclerotic disease in patients with newly diagnosed depression.

METHODS

This study used whole blood aggregometry, flow cytometry and ELISA to investigate platelet CD62P (P-selectin) expression and atherosclerotic markers (CD40, CD40L) as well as serum platelet factor 4 (PF-4) and beta-thromboglobulin (β-TG) levels in 46 participants. Patients with newly diagnosed, but not yet medically treated depression (n = 21) were compared to healthy control patients.

RESULTS

The platelet activation marker CD62P was significantly higher in patients with depression (2.62% depression versus 1.27% controls; p = 0.006). Further we found basal CD40 (6.7% vs. 4.8%; p = 0.002) and basal CD40L (31.0% vs. 22.0%; p = 0.025) to be elevated in patients with depression as compared to control persons. In addition sCD40L (52.7 vs. 44.4 ng/ml; p = 0.023) and β-TG differed significantly in depressed patients (206.9 vs. 182.8 ng/ml; p = 0.001). However, basal CD41 (97.0% vs. 96.3%; p = 0.57), CD42b (96.7% vs. 94.7%; p = 0.28) and PF-4 (89.61 vs. 81.75 IU/ml; p = 0.10) and the aggregometry results did not differ significantly between the study groups.

CONCLUSIONS

Our findings with elevated CD40 and CD40L as well as CD62P and β-TG in newly diagnosed patients emphasize that depression is linked to a prothrombotic and proinflammatory state and this possibly contributes to accelerated atherosclerosis.

BACKGROUND

Platelet activation in circulation is considered to be associated with thrombosis and inflammation; thus, sensitive and easy-to-use markers are necessary. In this study, we established a simple and rapid protocol to clinically examine leukocyte-platelet aggregate formation associated with activated platelets in circulation.

METHODS

Whole blood was stained with PC5-conjugated anti-CD45 monoclonal antibody and fluorescent isothiocyanate-conjugated anti-CD41 monoclonal antibody for leukocyte-platelet aggregate analysis. For platelet activation, 5 μm thrombin receptor-activated peptide (TRAP) or 2 μg/mL collagen was added. Samples were analyzed by EPICS XL (Beckman Coulter, Miami, FL, USA). Monocytes, neutrophils, and lymphocytes were gated based on differences in CD45 fluorescence intensity and side scatter. For each gate, the percentage (%) of platelets expressing CD41 was analyzed. Same drawing sample was stained with anti-CD62P monoclonal antibody. Platelet CD62P expression was then analyzed with gating for platelet cell population.

RESULTS

We analyzed leukocyte-platelet aggregates and platelet CD62P expression in 18 healthy individuals. Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, increased when platelets were activated by platelet agonists. Monocyte-platelet aggregates and neutrophil-platelet aggregates also increased over time with mild platelet activation.

CONCLUSIONS

Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, appear to be a sensitive marker of platelet activation in circulation.

Although the platelet activation profile after stroke is a well-known issue, the platelet reactivity assessed prospectively after ischaemic stroke still remains equivocal. The aim of this study was to evaluate the reactivity of platelets in response to stimulation with thrombin receptor-activating peptide (TRAP) at 1, 10 and 90 days after ischaemic stroke and to compare it with results obtained in control groups. We determined the increment in surface expression of CD62P, CD40L and monocyte- and granulocyte-platelet aggregate formation using five-colour flow cytometry in 86 subjects after an ischaemic event, in 62 disease controls, and in 38 healthy volunteers. We assessed the plasma levels of CD62P and CD40L soluble forms. In patients after stroke a significantly lower increment in CD62P surface expression (p < 0.01) and higher increments in both CD40L platelet surface expression (p < 0.01) and monocyte-platelet aggregate percentage (p < 0.01) were found at every studied time point, as compared with the control groups. Plasma levels of soluble CD62P (sCD62P) and soluble CD40L (sCD40L) were increased in stroke subjects in both the acute and the subacute phase of the stroke and they dropped to levels observed in controls at day 90 after the ischaemic incident. In all studied groups a positive correlation was noted between plasma levels of sCD62P and sCD40L. In conclusion, while at 3-month follow-up the levels of soluble forms normalize in stroke patients, the profile of platelet reactivity in response to activation with TRAP differs from that observed in the controls despite the secondary stroke prevention.

Previous studies have demonstrated that both cytomegalovirus (CMV) infection and prolonged cold ischemia of the allograft (CI) are associated with chronic rejection of renal transplants. The purpose of this study is to investigate the effect of CMV infection, of CI and of the combination of both, on the progression of chronic rejection, and to obtain a more detailed insight in their effects on the expression of adhesion molecules. Therefore, a rat transplantation model was used. Lewis recipients of renal allografts (with and without CI) from MHC-incompatible Brown Norway rats were inoculated with rat CMV or left uninfected. CMV infection alone resulted in an increased influx of CD4+ cells and macrophages early after infection, and in an increase in glomerular sclerosis and intima proliferation. CI caused an increase in infiltrating NK cells and an effect on intimal proliferation, glomerular sclerosis, and tubular atrophy. When CMV infection and CI were combined, an additive effect could be measured. This was however not the case for the function of the kidney. The creatinin showed a synergistic effect of the two influencing factors. Due to the CMV infection, an increase in CD49d cells was detected. CI resulted in an increase in CD18 cells and an increase in the expression of CD62P on vessels, and CD54 and CD44 on tubules. When CMV infection and CI were combined, all the effects caused by CMV and CI alone were present in an additional way. The results of the present study suggest that special attention should be paid to the recipient of an ischemically injured graft when either the donor or the recipient is CMV-infected. The patterns seen in histology, the infiltration of leukocytes and the expression of adhesion molecules, suggest that CI and CMV infection both have an effect on rejection, but act by different mechanisms.

METHODS

Data on the quality of platelet concentrates (PC) produced by the buffy coat method and stored beyond 5 days in plasma are limited. We therefore evaluated the quality of PCs prepared by leucocyte depletion of whole blood (Terumo WBSP, n = 10) or a buffy coat pool (Pall Autostop, n = 10), and stored for 7 days in plasma by assessing platelet parameters and markers of platelet activation.

RESULTS

In both types of PC, levels of glucose decreased during storage but were not totally depleted >> 11 mM on day 7). In contrast, lactate levels increased on storage and was consistently < 20 mM throughout, with pH maintained at>> 6.8 in all units. Hypotonic shock response scores were>> 47% in all units at day 7. On day 1, markers of platelet activation were significantly higher in WBSP PC, but by day 7 were similar for percentage CD63+ and CD62P + (40%) with levels of platelet microparticles and annexin V binding two-fold higher in WBSP. The expression of CD61 did not alter during storage and the percentage of platelets expressing CD42b was>> 88% in all units on day 7. RANTES (Regulated on activation, normal, T-cell expressed and secreted) and TGFbeta released from platelets by day 7 was < 800 ng/ml and 90 ng/ml, respectively. C3a(desarg) increased throughout storage in both types of PC, but without a commensurate increase in the terminal complex SC5b-9 or activation of factor XII.

CONCLUSIONS

Our data indicates that the in vitro characteristics of PCs prepared using these methods is maintained over storage for 7 days in plasma and is not associated with significant deterioration of platelet function. ONE SENTENCE SUMMARY: In vitro function of platelet concentrates prepared by either filtration of whole blood, or pooled buffy coats.

Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me(2)SO) and stored for extended periods at -80°C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me(2)SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at -80°C. The cryopreserved platelet units (n=9) were rapidly thawed at 37°C, reconstituted in 50% SSP+/plasma and stored at 22°C. Platelet recovery and quality were examined 1 and 24h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me(2)SO and additive solution produces acceptable in vitro platelet quality.

Leukocytes enter specific ovarian areas at a precisely defined moment, influencing cyclically changing structures such as follicles and corpora lutea. As yet, no studies have been published on the trafficking mechanisms involving the interaction between adhesion molecules on endothelial cells (ECs) and those on leukocytes. First, antibodies against human adhesion molecules were examined by flow cytometry with the aim of identifying the same bovine antigen. Western blot analysis and immunoprecipitation revealed that the molecules had the same molecular weight as their human counterparts. Afterwards, we investigated the distribution of these antigens in various ovarian stages using immunohistology. Among the molecules, P-selectin (CD62P) and L-selectin (CD62L) showed stage-dependent expression, and were thus examined further. In the preovulatory follicle, microvascular ECs were negative for CD62P. Few of the leukocytes expressed CD62L. In a freshly ruptured follicle, CD62P expression was found in the dilated vessels of the former thecal layer. Simultaneously, a large proportion of the rapidly increased numbers of leukocytes, mainly eosinophils, located around the microvessels of the outer thecal layer expressed CD62L. In the early corpus luteum development stage, CD62L showed peak expression with 70%-80% positive cells compared to leukocytes. In the secretory stage, the septal venules showed a consistent, but now weak, staining for CD62P. Few leukocytes expressed CD62L. During regression, the total number of leukocytes, now representing macrophages, increased significantly, but the proportion of CD62L-positive cells remained constant. In summary, we found a strong correlation of CD62P expression on activated ECs and the appearance of CD62L-positive leukocytes in the early corpus luteum development stage, suggesting the participation of both selectins in the migration of eosinophils under physiological conditions.

OBJECTIVE

To investigate the platelet activity and function in pregnancy induced hypertension (PIH) and gestational diabetes mellitus (GDM).

METHODS

Twenty-one patients with GDM and 23 patients with PIH in third-trimester were included. Twenty normal pregnant women in third-trimester served as controls. Platelet count (PC), mean platelet volume (MPV) were determined on Cell-DYN 1600 and the expression of CD62P was analyzed on FACSC alibur.

RESULTS

(1) PC was (181 +/- 56) x 10(9)/L in PIH, (206 +/- 60) x 10(9)/L in GDM and (229 +/- 56) x 10(9)/L in controls, respectively. PC in PIH was lower than that of controls (P < 0.01), but there was no significant difference between GDM and controls. (2) MPV was (11.2 +/- 2.0) fl in PIH, significantly higher than that of controls (8.7 +/- 1.6) fl (P < 0.001). In GDM, MPV was (9.5 +/- 1.6) fl, without significant difference compared with that of controls. (3) The expression of CD62P increased significantly in PIH compared with controls [CD62P: (42 +/- 13)% vs (26 +/- 7)%, P < 0.001; CD62P(I): 109 +/- 39 vs 75 +/- 13, P < 0.01]. In GDM, the expression of CD62P also increased significantly compared with the normal pregnancy [CD62P(%): (42 +/- 14)% vs (26 +/- 7), P < 0.001; CD62P(I): 100 +/- 42 vs 75 +/- 13, P < 0.05]. (4) All parameters had no significant difference between PIH and GDM.

CONCLUSIONS

Platelet activity is enhanced in PIH and GDM. It may play an important role in the pathogenesis and development of the two diseases.

Haemorrhage is often responsible for the lethal course of acute myeloid leukaemia (AML). Previously, multiple platelet function defects were identified by flow cytometric analysis of platelet activation markers in AML. The role of flow cytometric analysis of platelet function in characterization of prognostic markers of haemorrhage in AML patients has not been well elucidated. The objective of this prospective study was to analyse platelet function in 50 AML patients at diagnosis and to compare results with clinical bleeding score, graded by common toxicity criteria. Platelet activation markers CD62P, CD42b, CD63 and PAC-1 were analysed following in vitro activation by thrombin receptor activating peptide. The following plasma haemostasis parameters were measured: soluble P-selectin, activated partial thromboplastin time, thrombin time, prothrombin time, D-dimer, fibrinogen, and von Willebrand factor antigen. In a multivariate analysis, P-selectin (CD62P) <36 molecules of equivalent soluble fluorochrome x 10(3) (P < 0.0015) and platelet count <40 x 10(9)/l (P = 0.01) were significant predictors of haemorrhage at diagnosis. Haemorrhage at diagnosis predicted grade 3-4 haemorrhage in the first 28 d following diagnosis (P = 0.018). The presented results indicate that low P-selectin is a prognostic marker of haemorrhage in AML.

OBJECTIVE

To explore the effects of Tongxinluo Capsule (TXLC) on platelet activating factor, vascular inflammation factor and vascular endothelial function in patients with essential hypertension (EH) complicated with diabetes mellitus (DM).

METHODS

One hundred patients of EH with DM were equally assigned to the TXLC group (treated by TXLC) and the control group (treated with the conventional therapy). Their fasting blood drawn from the cubital vein on the next morning of hospitalization was taken for determining serum level of high sensitivity C-reactive protein (hs-CRP) by emulsion immunoenhancement turbidimetry; plasmal fibrinogen C (FIB-C) by diffusive turbidimetry; platelet activating indices, CD62p and glucose protein (GP) II b/III a receptor complex by flow cytometry; endothelin-1 (ET-1) by radioimmunoassay and nitrogen oxide (NO) content by enzyme method. The outcomes were compared with those of 50 healthy persons. After patients were treated for 8 weeks, all the above-mentioned indices were reexamined and compared between groups. Results Blood levels of hs-CRP, FIB-C, CD62p, GP II b/IIIa and ET-1 in patients were significantly higher than those in healthy persons (all P < 0.01). All the indices as well as the blood pressure (both systolic and diastolic) reduced in patients of both groups significantly (P < 0.05 or P < 0.01), but the reducing was more significant in the TXLC group than in the control group. Besides, level of NO significantly increased in the TXLC group (P < 0.05).

CONCLUSIONS

TXLC can inhibit the platelet activation and vascular inflammation response, also improve the vascular endothelial function in patients with EH complicated with DM. It may play a certain role in preventing and treatment of the occurrence of thrombotic complications in them.

BACKGROUND

Umbilical cord blood (CB) has been used as an alternative source for haematopoietic stem cell transplantation (HSCT) in recent years. However, delayed platelet recovery is frequently associated with CB HSCT. Megakaryocytes (Mk) are the specialised precursors of platelets and they are among the rarest haemopoietic cell types. Despite the rapid expansion of our knowledge of megakaryopoiesis in recent years, many questions, such as the molecular regulatory mechanisms in Mk differentiation and maturation, platelet formation and release, remain unanswered in CB-derived megakaryopoiesis. Variations can be seen from the literature by individual investigators using different approaches for Mk-specific differentiation and maturation induction. The development of in vitro culture methods to obtain sufficient numbers of Mks from readily available haematopoietic stem cells is of value for both basic research and clinical applications.

METHODS

The CD34+ cells from cord blood samples were cultured in serum-free medium with haematopoietic growth factors (GFs), such as IL-3, stem cell factor (SCF), and thrombopoietin (Tpo). The differentiation of Mk was monitored using Mk- and platelet-specific monoclonal antibodies and flow cytometric analysis. The morphology of the cultured cells was studied by both light and electronic microscopy (LM and EM). The involvement of the human Notch gene family members was studied by real time-polymerase chain reaction (RT-PCR). Maturation of the cultured Mks was studied using flow cytometric analysis for both platelet-specific surface markers and enodomitosis. Platelet activation was assessed in the cytoplasmic fragments harvested from the cultures.

RESULTS

Specific Mk differentiation of >70% resulted from a 2-step culture approach using IL-3, SCF and Tpo for 7 days followed by Tpo only for another 14 days. RT-PCR showed high-level expression of both Notch-1 and its ligand, Jagged-1, in the cultured Mks. Limited levels of polyploidy (>4N, endomitosis, EnM) were observed in the cultured Mks. The results also showed that the cytoplasmic fragments from the cultures responded to platelet activation reagents, including ADP and collagen, marked by upregulation of platelet-specific activation markers, such as CD62P (P-selectin) and PAC-1 (gpalphaIIbbeta3).

CONCLUSIONS

The methods used in this study are specific for differentiation of Mk from CB CD34+ cell, which can partially mature and produce functional platelets in vitro. This approach for human Mk differentiation could be further optimised and may be adapted on larger scales for clinical purposes.

Nitric oxide (NO) is known as a regulator of platelet function by its anti-adhesive, anti-aggregating, and disaggregating properties. We investigated the modulating effects of the NO-releasing compound SIN-1 (3-morpholino-sydnonimine) on platelet surface glycoprotein (GP) expression during stimulation with human alpha-thrombin. Analysis was performed with two-color flow cytometry using fluoresceine-isothiocyanate (FITC) and phycoerythrin-(PE)-conjugated monoclonal antibodies (MoAbs) directed against GPIb CD42b), GP IIb-IIIa (CD41), P-selectin (CD62P), and MoAb PAC-1 directed against activated GP IIb-IIIa. Preincubation of platelets with SIN-1 (IC50: 1 microM) significantly decreased expression of both total and activated GP IIb-IIIa, and P-selectin in platelets stimulated with thrombin (ED50: 0.05 U/ml), whereas thrombin-induced downregulation of GP Ib was not attenuated. P-selectin expression increased in thrombin-stimulated platelets over time; in contrast, activated GP-IIb-IIIa decreased after an initial peak, indicating that thrombin-induced GP IIb-IIIa activation is spontaneously reversible. SIN-1 reduced P-selectin expression only when added before or at the same time as thrombin, whereas conformationally changed GP-IIb-IIIa was significantly reversed at up to 60 minutes after stimulation by SIN-1. In conclusion, NO attenuates activation marker expression in a dose and time dependent manner. GP-IIb-IIIa is highly sensitive to NO which not only prevents receptor activation but also promotes reversal of activated GP IIb-IIIa complex.