The efficiency of in vitro platelet production is considerably low compared with physiological activity due to the lack of pivotal factors that are essential in vivo. We developed an ex vivo platelet production system, introducing human megakaryocytes into an isolated porcine thighbone and culturing in closed circuit. The efficiency of the ex vivo platelet production system was compared to those in vivo and in vitro. CD61+ platelet-like cells were counted by immunostaining and flow cytometry. Results showed that 4.41 ± 0.27 × 103 CD61+ platelet-like cells were produced by 1 × 103 megakaryocytes in the ex vivo system, while 3.80 ± 0.87 × 103 and 0.12 ± 0.02 × 103 were produced in the in vivo and in vitro systems, respectively. Notably, ex vivo and in vitro production systems generated cells that responded well to thrombin stimulation and expressed functional molecules, such as CD62P. Overall, our ex vivo production system was comparable to in vivo production system and produced platelet-like cells that were functionally superior to those produced in vitro. In future, the present ex vivo production system implementing xenogeneic bone marrow would offer a promising alternative for industrial-scale production of platelet-like cells.

BACKGROUND

Recovery and survival of transfused platelets (PLTs) are usually assessed by radioisotope labeling methods for evaluation of transfusion efficacy and new progress in the processing of PLT concentrates. Alternative, nonradioactive methods are warranted.

METHODS

A multicolor flow cytometry method was developed for simultaneous studies of recovery, survival, and function of transfused PLTs. Eight consecutive patients undergoing allogeneic stem cell transplantation (TX) were transfused with apheresis PLTs of nonself human leukocyte antigen (HLA) Class I types, and HLA Class I discrepancy between donor and recipient was used to identify transfused PLTs. Hematologic status and HLA Class I surface expression were analyzed immediately before transfusion, 1 and 6 hours after transfusion, and daily during the subsequent week. PLT activation was assessed by surface expression of CD63, CD62P, or CD42a, before and after stimulation with thrombin receptor agonist peptide.

RESULTS

PLT recovery was 43, 41, and 31% for fresh (5-72 hr old) and 30, 27, and 17% for stored (73-148 hr old) PLTs, after 1, 6, and 15 to 28 hours, respectively. Survival of fresh versus stored PLTs were 160 and 105 hours, respectively. Spontaneous PLT activation and residual activation potential were almost equal for fresh and stored PLTs. PLT engraftment was detected between Day 7 and Day 9, which was significantly earlier than first sign of neutrophil engraftment (Days 11-19; p=0.01).

CONCLUSIONS

Flow cytometry is an attractive alternative to radiolabeling of PLTs for simultaneous studies of survival, recovery, and function of transfused PLTs and early detection of PLT engraftment after allogeneic stem cell TX.

BACKGROUND

After severe polytrauma the dynamic process of coagulation may deteriorate towards a trauma-induced coagulopathy (TIC) promoting a dramatic increase in morbidity and mortality. Recent evidence suggests that microparticles (MPs) play a pivotal role at the interface between cellular and plasmatic coagulation systems. However, the impact of MPs on functional coagulation has not been clarified yet in the setting of traumatic injuries. We assessed the temporal patterns of circulating MP concentrations including their cellular origin in the context of clinical presentation and global coagulation assays.

METHODS

Blood samples from 22 consecutive polytrauma patients (ISS ≥16) from 2015 were collected at hospital admission, after 24 and 72 h and compared to those from healthy individuals and minor injured patients with isolated extremity fractures. Flow cytometry (BD Accuri C6; Heidelberg/Germany) was used to determine MP concentrations and cellular origin using cell-specific markers (platelet derived (PDMP): CD42b+, CD61+, CD62p+; endothelial cell derived (EDMP): CD144+, CD62e+, CD144+/62e+). Results were correlated with clinical data and results from viscoelastic testing (ROTEM).

RESULTS

Twenty two polytrauma patients (17 males, agemedian 60 yrs) with a median ISS 26.5 (IQR 14.5) were assessed. PDMP and EDMP concentrations increased significantly in polytrauma patients as compared to healthy individuals and minor injured patients. MP concentrations correlated with injury severity (CD144+: ρsp = 0.79, p < 0.001; CD42b+: ρsp = 0.61, p < 0.001). EDMP displayed a negative correlation with aPTT (CD144/62e+, ρsp = - 0.55, p < 0.05), INR (CD144/62e+, ρsp = - 0.61, p < 0.05) and ROTEM-INTEM CT (CD144/62e+, ρsp = - 0.68, p < 0.05) reflecting increased dynamics of clot formation and an overall procoagulative effect. Additionally, EDMP showed a negative association with FIBTEM values (10 min amplitude, maximum clot firmness) indicating a fibrinolytic potential.

CONCLUSIONS

In a small cohort, analysing most severly injured patients, the association of increased MP levels and altered coagulation parameters could be demonstrated. However, these findings are based on correlation analysis, which do not enable causel evidence. Therefore, further in-vitro studies are needed analysing the underlying pathomechanisms.

CONCLUSIONS

In conclusion, this study could demonstrate that PDMP and EDMP levels increase significantly following polytrauma correlating with injury severity. Although severe coagulopathy was not observed, EDMP levels were associated with improved coagulation parameters suggesting their essential role for regulating blood coagulation after trauma.

OBJECTIVE

Platelet function is an important factor for the fate of intensive care patients. Several factors may influence this function. Recently, it was demonstrated that hydrocortisone has immunologic effects in septic shock and therefore may affect cell adhesion molecules. The aim of the present study was to examine effects of hydrocortisone on platelet receptor expression in healthy individuals and septic patients in vitro.

METHODS

Citrated blood samples were drawn from 10 healthy volunteers and 10 septic patients. Samples were adjusted with hydrocortisone to final concentrations of 4.5 microg mL(-1), 9.0 microg mL(-1) (sepsis-equivalent bolus) and 90 microg mL(-1), respectively. A control group received no additional hydrocortisone. Expression of CD62P, CD41, PAC-1 and CD42b on the surface of resting or agonist-stimulated platelets was determined by whole blood flow cytometry using fluorescence-labeled monoclonal antibodies.

RESULTS

Hydrocortisone had no significant influence on the expression of CD62P, CD41 and PAC-1. After administration of 90 microg mL(-1) hydrocortisone the expression of CD42b was decreased compared to controls after activation. Differences between volunteers and sepsis patients were found for all receptors after activation.

CONCLUSIONS

Hydrocortisone mediates immunmodulating effects in therapy of patients suffering of septic shock without involvement of specific platelet receptors in vitro.

Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and < 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.

OBJECTIVE

The effects of the volatile anaesthetic desflurane on platelet activation in vitro were studied and compared to those of halothane.

METHODS

Platelet-rich plasma was exposed to 2 MAC of desflurane or halothane, or air only and stimulated by platelet agonists ADP (2.5, 5 and 10 micromol L(-10) and collagen (10 microg m(L-1)). Platelet response was measured by Born aggregometry (maximum aggregation response, area under the curve) and flow cytometry (mean channel fluorescence, percentage of CD62P-positive cells, index of platelet activation for positive platelets).

RESULTS

Aggregation response was significantly reduced in platelets exposed to desflurane or halothane; the inhibitory effect was more pronounced when the areas under the curve were analysed: values ranged from 37.5% to 73.3% of control samples for ADP stimulation and 77.1% to 79.8% for collagen stimulation. CD62P expression before and after stimulation with receptor agonists was not statistically different in platelets exposed to desflurane, halothane or air.

CONCLUSIONS

By impairing platelet aggregation while not affecting alpha-degranulation desflurane has a differential effect on various aspects of platelet activation similar to halothane. Our results are compatible with the hypothesis of an impairment of platelet thromboxane receptor signalling by halothane. We suggest a similar mechanism for desflurane.

Platelet derived microvesicles, which are shed from platelets upon platelet activation, interact with monocytes in the blood. In this study the nature of this interaction was characterized in a model system with the monocytic cell line MM6 and isolated platelet derived microvesicles (PMV). The interaction of PMV with MM6 is separated in two consecutive steps, which are partially overlapped in time. In a first step there is an immediate conjugate formation with single MM6 and PMV, which was proved microscopically and by cytometry measurements. This process is dependent on CD62P, determined by an inhibition after pre-incubation with anti-CD62P. After a lag time of 4 min this process is supplemented by an aggregate formation of single conjugates, which leads finally to one macroscopic visible aggregate. The Nature of this aggregate was characterized by immunohistochemistry and laser aggregatometry. An addition of GPRP blocks the formation of a fibrin network and also the aggregate formation, proving the necessity of fibrin network formation. This was also shown by diminishing the aggregate formation by addition of hirudin. Finally fluorescent microscopic images proved the necessity of a fibrin network holding MM6 cell/PMV aggregates together. Even pure PMV can form such an aggregate only visible as thin film and less stable as the cell PMV aggregate. The described process might be important in vivo causing thrombotic events without direct involvement of platelets. Especially in situations with extreme PMV levels, such as acute coronary heart disease, trauma and sepsis, these events could lead to the appearance of haemostatic complications.

Platelet storage lesion is characterized by morphological changes and impaired platelet function. The collection method and storage medium may influence the magnitude of the storage lesion. The aim of this study was to compare the newly introduced interim platelet unit (IPU) platelet concentrates (PCs) (additive solution SSP+, 40% residual plasma content) with the more established buffy-coat PCs (SSP, 20% residual plasma content) and apheresis PCs (autologous plasma) in terms of platelet storage lesions. Thirty PCs (n=10 for each type) were assessed by measuring metabolic parameters (lactate, glucose, and pH), platelet activation markers, and in vitro platelet aggregability on days 1, 4, and 7 after donation. The expression of platelet activation markers CD62p (P-selectin), CD63 (LAMP-3), and phosphatidylserine was measured using flow cytometry and in vitro aggregability was measured with multiple electrode aggregometry. Higher platelet activation and lower in vitro aggregability was observed in IPU than in buffy-coat PCs on day 1 after donation. In contrast, metabolic parameters, expression of platelet activation markers, and in vitro aggregability were better maintained in IPU than in buffy-coat PCs at the end of the storage period. Compared to apheresis PCs, IPU PCs had higher expression of activation markers and lower in vitro aggregability throughout storage. In conclusion, the results indicate that there are significant differences in platelet storage lesions between IPU, buffy-coat, and apheresis PCs. The quality of IPU PCs appears to be at least comparable to buffy-coat preparations. Further studies are required to distinguish the effect of the preparation methods from storage conditions.

BACKGROUND

The INTERCEPT Blood System for Platelets (PLT) utilizes amotosalen (S-59) in combination with ultraviolet A (UVA) light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate PLT concentrates. However, limited data are available on the quality of INTERCEPT-treated double-dose (DD) buffy-coat (BC) PLT units allowing a single treatment procedure to produce two pathogen-inactivated PLT units for transfusion.

METHODS

The objective of this study was to evaluate potential in vitro effects of the INTERCEPT treatment on pools of 7 BCs as compared to untreated units. Functional, phenotypic and mitochondrial properties of DD BC PLTs during storage over 7 days were studied.

RESULTS

For some parameters measured, small yet significant differences were observed including PLT count (P < 0·05), pH, pCO2 and glucose concentration. Throughout storage, no significant differences were observed in ATP levels, ESC, HSR reactivity and CD62P expression. Similarly, no differences were observed in the expression of PAC-1, CD42b and PECAM-1 at any time-points. The mitochondrial membrane potential (MMP) determined by JC-1-labelling was well maintained until day 7 in all treated and untreated units (>90%). The release of sCD40L increased over time (P < 0·01) in all units but without any significant differences between treated and untreated PLTs.

CONCLUSIONS

Our data demonstrate that photochemical pathogen inactivation of DD-BC PLT concentrates with the INTERCEPT Blood System had no influence on the PLT in vitro quality over the 7 day of storage. However, whether in vivo efficacy of INTERCEPT-treated PLTs is affected may require clinical evaluation.

BACKGROUND

This study was purposed to establish reference values for platelet activation markers and leukocyte-platelet aggregates in the evaluation of the platelet function tests using flow cytometry.

METHODS

Whole blood samples were obtained from 30 volunteers of healthy adults. Diluted blood samples, either in the resting state or in activated state by the addition of agonist, 20 microM ADP or 100 microM TRAP, were stained with fluorescent conjugated monoclonal antibody of PAC1 or CD62P. Then, the percentages of expression for each marker were analyzed by flow cytometry. For leukocyte-platelet aggregates, monoclonal antibodies of CD41a, CD14 and CD45 were added simultaneously to undiluted whole blood.

RESULTS

Reference values for the percentages of the expression of PAC1 and CD62P, respectively, were 0.1-12.5% and 0.0-4.7% at the resting state, 65.3-92.4% and 39.0-75.7% with the addition of 20 microM ADP, and 68.1-93.1% and 60.5-91.2% with the addition of 100 microM TRAP. Reference values for leukocyte-platelet aggregates, granulocyte-platelet aggregates, and lymphocyte-platelets aggregates were 2.8-23.6%, 5.3-34.2%, and 4.9-21.6%, respectively.

CONCLUSIONS

The platelet activation markers at the resting or an activated state with agonists and leukocyte-platelet aggregates could be analyzed using flow cytometry. These reference values should be helpful in interpreting platelet function tests by flow cytometry.

BACKGROUND

During preparation and storage of apheresis concentrates, platelets are being activated. One of the alterations that occur during this process is an increased expression of P-selectin (CD62p) on the cytoplasmic surface of platelets. This neoepitope represents a ligand for the binding of platelets to WBCs. It has been suggested that the activation of platelets is associated with the sequestration of platelets after transfusion. In this in vivo study, the binding of platelets to WBCs was analyzed following transfusion of platelet concentrates (PCs).

METHODS

Double apheresis concentrates were prepared with two different cell separators. One of the split products was stored for 1 to 2 days and the other one for 3 to 5 days. Flow cytometry was applied to analyze the degree of platelet activation in vitro, and also to measure the extent of platelet binding to WBC subclasses in vivo after transfusion into patients.

RESULTS

The results of this study show that platelet activation occurs during apheresis and storage of PCs. After transfusion of the PCs, no significant binding of platelets to T or B-cells could be detected. However, a significant binding of platelets to monocytes and neutrophil granulocytes occurs. While in Baxter PCs stored for 1-2 days the amount of platelet-leukocyte aggregates in vivo was higher compared to COBE PCs, no such difference could be detected anymore for the PCs stored for 3-5 days.

CONCLUSIONS

This study demonstrates that binding of activated platelets occurs to monocytes and neutrophil granulocytes but not to T- and B-cells in the circulation after transfusion. In addition, the interaction of platelets and WBCs is dependent on the degree of P-selectin expression. Platelets showing a higher degree of activation adhere to WBCs to a higher degree than nonactivated platelets.

The paper reviews literature updates on the organization and specific features of megakaryocytopoiesis, the mechanisms of its regulation, possible ways for platelets to occur from megakaryocytes. It characterizes thrombocytopoiesis, by describing the detailed structural organization of platelets and gives data on the relationship of the morphofunctional features of platelets to the ploidy of megakaryocytes that give rise to the latter. The structure and clinical value of a number of platelet activation markers, such as CD41/CD61, CD42, CD62p, platelet-leukocyte aggregates, microvesicles, are described.

OBJECTIVE

To explore platelet activation and the protective effect of aprotinin in patients with hepatolithiasis.

METHODS

The count of platelets and levels of CD62P and CD63 were measured by flow cytometry in 38 patients with hepatolithiasis. Several measurements were carried out after treatment with aprotinin.

RESULTS

The levels of CD62P, CD63 in patients with hepatolithiasis were higher than those in patients with cholecystolithiasis (P<0.05), but the count of platelets was lower (P<0.05). After operation, the levels of CD62P, CD63 were significantly increased in patients with hepatolithiasis, but the count of platelets was lower (P<0.05). Postoperative levels of CD62P, CD63 were significantly lower in patients treated with aprotinin than in normal controls (P<0.05); but there was no significant change in the count of platelets in the two groups.

CONCLUSIONS

Platelet activation occurs in patients with hepatolithiasis, and may be inhibited by aprotinin.

Clinical screening tests for blood coagulation that use plasma as samples cannot estimate the participation of platelets, leukocytes, and erythrocytes in blood coagulation system. We developed an assay to evaluate the total coagulation ability of blood and whole blood prothrombin time (WPT) using the principle of prothrombin time with the diluted-tissue factor as a trigger and a newly developed apparatus, STA, viscosity change detection system (electromagnetic clot detection). The activation of platelets by Ca ionophore shortened WPT and increased the expression of CD62P on the platelet surface. WPTs in citrated blood of spontaneous hypertensive rats (SHR) were significantly shorter than those of controls, Wistar Kyoto rats (WKY). Plasma levels of thrombin-antithrombin III (TAT) in SHR were significantly higher than those of WKY. Moreover, WPT and TAT levels were significantly correlated. Based on these results, WPT was found to be useful to estimate the activation of blood coagulation in whole blood.

Platelet-leukocyte conjugates are increased in cardiovascular disease, but exercise is also able to trigger platelet-leukocyte formation in healthy subjects. The aim was to investigate the heterogeneity of platelet-leukocyte conjugate formations triggered by short term exercise. 18 healthy non-smokers underwent a 90 second maximal test on a SRM cycle ergometry system and a control experiment. Blood samples were taken after 30 min rest, immediately before and after, 15 min and 1 h after exercise. The different platelet-leukocyte conjugates were detected by flow cytometry via CD45, CD14, CD16, CD41, together with CD62P antibodies for the investigation of platelet activation in the conjugates. In addition, a stimulation of conjugate formation in vitro with 8 microM TRAP-6 was initiated. Immediately after exercise platelet-granulocyte (+24%), and -lymphocyte (+17%) conjugates were increased (p<0.01), while the platelet-monocyte conjugates (+40%) were enhanced (p<0.05) 15 min after exercise. The differentiation after stimulation showed that the regular (CD14(+)16(-); +32%) and mature (CD14(+)16(+); +35%) monocytes were both increased after exercise (p<0.01) but the regular monocytes were preferred (p<0.001) in platelet-monocyte conjugate formation. In addition, these conjugates revealed the highest CD62P expression. Maximal short term exercise is useful for the investigation of platelet-leukocyte formation; e.g., it could be shown, that regular monocytes may be preferred in conjugate formation and that these conjugates revealed the highest CD62P expression.

We hypothesized that mean platelet volume (MPV), a reliable marker of platelet activation, might be elevated in primary Raynaud's phenomenon (PRP) even if there was no thrombotic complication in our subjects. In this retrospective-cohort study, we examined the clinical value of MPV in 200 patients with PRP and 116 clinical controls, and measured MPV and platelet P-selectin (CD62P) in all study participants. We also evaluated the effect of age, gender, and disease duration on these platelet activation markers in PRP. MPV and CD62 positivities were significantly (p<0.001) elevated in patients with PRP compared with controls. These differences retained when patients and controls were analyzed according to age, gender, and the disease duration. In logistic regression analysis, MPV (OR: 15.8, 95% CI: 8.14-30.64, p<0.001) and CD62P (OR: 11.3, 95% CI: 4.85-26.12, p<0.001) were found to be independently associated with PRP. In conclusion, increased MPV is independently related to PRP, and its level was not influenced by age, gender, and the duration of PRP.

OBJECTIVE

To assess the effects of hormone replacement therapy (HRT) on platelet activation in postmenopausal women compared with premenopausal women.

METHODS

The expressions of CD41 and CD62P in fifteen postmenopausal women before and after HRT were detected using flow cytometry (FCM), with fifteen premenopausal women with a mean age of 47 years as controls.

RESULTS

The expressions of CD41 and CD62P in postmenopausal women were higher than those in the control group. CD62P(%), CD62P(I) and CD41 were reduced from 36.40 +/- 5.9, 37.75 +/- 5.8, and 470.11 +/- 74.0 to 27.97 +/- 5.6, 26.64 +/- 4.9, and 303.23 +/- 72.8 after six months of HRT (P < 0.05).

CONCLUSIONS

Platelet activation in postmenopausal women was higher than in premenopausal women and was reduced significantly after six months of HRT. HRT may have a favorable effect on reduction of platelet activity.

The extent of platelet activation after exhaustive exercise remains under discussion. Previous studies have provided contrary data, probably due to differences in the methodologies and the enrolled subjects. In the present study a maximal treadmill exercise (TR) was used to test platelet activity and -reactivity in 13 healthy non-smoking men. Blood samples were taken after a 30min rest, immediately before and after exercise, and 1h after completion of exercise. Platelets were analysed by whole blood flow cytometry either directly or after in vitro stimulation by incubating the blood samples for 10min with TRAP-6 (10µM) or ADP (5µM or 2,5µM). Binding of an anti-CD62P antibody or a PAC1 antibody directed against the activated fibrinogen receptor GPIIb/IIIa were used as a measure of platelet activation. Immediately after TR the percent CD62P positive platelets (%PC) unstimulated increased (p<0.01) from 0.77±0.06 to 1.12± 0.09 %PC and in PAC1 (p<0.05) from 2.32 ±0.54 to 3.83±0.81 %PC (mean±SEM). After ADP-stimulation an increase from 4.18±1.02 to 5.69±1.40 %PC in CD62P (p<0.01) and from 45.7±3.4 to 57.9±6.6 %PC in PAC1 (p<0.05) after TR were detected. Using TRAP-6-stimulation only the increase of PAC1 (p<0.01) after TR was different in comparison with the control experiment without exercise. Soluble CD62P in plasma as a marker of platelet and endothelial cell activation was also enhanced (p<0.05) after TR. Although these results indicate that exhaustive exercise lead to a small platelet activation and an increase in platelet reactivity, it is rather doubtful that these changes alone implicate a prothrombotic situation in healthy young non-smokers.

OBJECTIVE

Placental fibrin deposits in patients wih recurrent spontaneous abortion (RSA) indicate an exaggerated haemostatic response. This 'hypercoagulability' may involve pro-coagulant factors such as circulating microparticles (MPs). We investigated the relationship between circulating pro-coagulant MPs and systemic coagulation in RSA patients.

METHODS

Platelet- and endothelial cell-derived microparticles (PMPs, EMPs) were evaluated by flow cytometry in RSA patients (n = 51) and compared to controls (n = 24) using annexin V (total numbers of MP), and antibodies against CD61, CD63 and CD62P (PMP), as well as CD144 and CD62E (EMP). Prothrombin fragment 1 + 2 (F(1+2)) and thrombin generation were determined to assess the pro-coagulant potential of MP.

RESULTS

Numbers of annexin V-binding MP were nearly similar in RSA patients and controls. However, a subgroup of ten RSA patients (10/51; 20%) presented with MP concentrations >10,000 x 10(6)/L, compared to only one women out of the control group (1/24; 4%; P = 0.038). Neither PMP and EMP nor F(1+2) and thrombin generation differed significantly within the study population.

CONCLUSIONS

The present study shows that circulating MPs are not directly associated with the extent of systemic coagulation activation in RSA patients. We hypothesize that increased numbers of circulating MPs either are only indirectly associated with coagulation during pregnancy of RSA patients, or affect abortion via mechanisms independently from hypercoagulation.

BACKGROUND

The effects of high intensity interval training (HIIT) on inflammatory markers and endothelial function have been extensively shown. However, the acute effect of HIIT on platelet activation and function in patients with recent revascularization is unclear.

OBJECTIVE

The purpose of present study was to compare the responses of platelet activation (CD62P) and function (platelet aggregation) to high intensity interval exercise (HIIE) and moderate continuous exercise (MCE) in coronary artery bypass grafting (CABG) and percutaneous coronary interventions (PCI) patients.

METHODS

Thirty patients who had CABG or PCI were randomly divided into HIIE, MCE and control groups. After determining the VO2peak, subjects in the MCE group carried out 30 min of continuous exercise at 60% of VO2peak, whereas, the subjects in HIIE group performed an interval protocol consisted of 8 repetitions of 2 min activity (running on treadmill) at 90% of VO2peak interspersed by 2 min of active recovery between repetitions at 30% of VO2peak . Subjects in control group were seated and had no activity for the same period of time. Two blood samples were collected before and immediately after exercise and were analyzed for markers of platelet activation and function.

RESULTS

Data analyzes revealed that increases in platelet aggregation induced by ADP and corrected for increases in platelet count in response to MCE trial was significantly lower than HIIE group (P < 0.05). In addition, responses of CD62P to MCE trial was significantly lower compared to HIIE group (P < 0.05). Changes in plateletcrit and platelet distribution width were significantly different among the three trials where the PCT and PDW following the HIIE were higher than MCE. Platelet count increased significantly (P < 0.05) by 13% following HIIE trial.

CONCLUSIONS

Based on the findings of the present study it could be concluded that the risk of exercise-induced thrombosis is higher during HIIE than MCE in patients with recent revascularization.

FVIII is an important cofactor in the tenase coagulation factor complex, lack of FVIII causes severe bleeding, whereas high FVIII levels seem to be associated with venous and arterial thromboembolism. Resting platelets do not bind FVIII, but activated platelets bind unactivated FVIII if vWF is not present. We investigated a possible influence of platelet bound FVIII on platelet function itself as it is unclear if there is a direct effect of FVIII on platelet function. The influence of FVIII on platelet function was investigated by flow cytometric analysis of P-selectin expression (CD62P) and PAC-1 binding before and after submaximal stimulation with TRAP-6 (5 microM final concentration), by confocal microscopy and by platelet aggregometry. For flow cytometry and confocal microscopy, washed platelets were incubated with human recombinant FVIII for 5 min at 37 degrees C. Analysis of platelet surface area was measured by computerized image analysis. Treatment with FVIII only caused no changes in P-selectin expression or PAC-1 binding, respectively. Stimulation of platelets with TRAP-6 increased the expression of P-selectin (445%) and PAC-1 binding (934%) as expected. These effects were further increased when platelets were stimulated with TRAP-6 and FVIII (P-selectin 499%, difference not significant; PAC-1 1626%, P < 0.05. Values were expressed in%, related to unstimulated, buffer treated platelets). Platelet spreading on fibrinogen was significantly increased when platelets were treated with FVIII and TRAP-6 compared to TRAP-6 alone (368 vs. 307 average pixel/platelet, P<0.05). In addition platelet aggregation was enhanced when platelets were stimulated with FVIII and TRAP-6 compared to TRAP-6 alone. FVIII can act as a positive regulator of platelet function in TRAP-co-stimulated platelets. We hypothesize that FVIII induced increase in platelet activation might contribute to venous and even arterial thrombus formation in patients with high FVIII levels.

There is still controversy about the correlation of thrombocytosis and thrombosis complication. Using a rodent splenectomy-induced thrombocytosis model and a thrombogenic endothelial damage model (inverted suture resulting in an intraluminal thrombogenic adventitia of divided femoral artery), the authors investigated whether reactive thrombocytosis with or without endothelial damage contributes to the patency of microvascular anastomosis. Four experimental groups were evaluated in this study: 1) sham operation without thrombogenic anastomosis after femoral artery division; 2) sham operation with thrombogenic anastomosis; 3) thrombocytosis alone without thrombogenic anastomosis; 4) thrombocytosis with thrombogenic anastomosis (each subgroup n = 10, total N = 40). Vascular patency was assessed after immediate operation and on the seventh day postoperatively. Platelet counts and platelet activation (CD62P) were studied in correlation to microvascular patency. In rats without thrombogenic anastomosis groups, there were no significant differences in CD62P expression on platelets (p = 0.09), the patency rates (p = 0.561), or perfusion units (p = 0.746) before and after arterial reanastomosis between rats with and without thrombocytosis, respectively. However, the thrombogenic anastomosis of femoral artery in thrombocytosis and control groups showed significantly increased CD62P expression (p < 0.05), decreased the perfusion unit (p < 0.05), and patency rate (p < 0.001), compared with rats without thrombogenic anastomosis of femoral artery in both groups. In summary, this study demonstrates that microvascular anastomosis can be performed safely with reactive thrombocytosis alone without thrombogenic anastomosis. Meticulous microvascular anastomosis without triggering platelet activation is the most important factor to prevent thrombosed vessels in microsurgical anastomosis.