Since the 19(th) century an association between cancer and thromboembolic events has been described, with a poorer survival prognosis. Production and secretion of procoagulant factors affect tumor biology and cancer-associated thrombosis. Tissue factor (TF) exerts coagulant and protease activated receptor (PAR)-dependent signaling effects, both of which can contribute to tumor progression. Tumor cells are also capable of shedding TF-positive microparticles, suggesting a contribution to cancer-associated thrombosis at a distance from the tumor. Selected tumors are capable of ectopically expressing FVII and/or FX, which may lead to increased procoagulant features of tumors. Alternatively spliced TF (asTF) may affect tumor progression by inducing tumor growth and angiogenesis in an integrin dependent manner. Ectopic thrombin production also affects tumor progression by influencing proliferation rate, angiogenesis, invasion and metastasis. However, the roles of these coagulation factors in tumor progression and cancer-associated thrombosis are still not fully understood. In this review we will discuss several coagulation factors and their contribution on cancer progression and venous thromboembolism.

Tissue factor (TF) antagonists targeting the factor VII (FVII) binding domain have been shown to interrupt acute vascular thrombus formation without impairing haemostasis in non-human primates. In this study, we evaluate whether a human/mouse chimeric monoclonal antibody (ALT-836, formerly known as Sunol-cH36) blocking the factor X/factor IX (FX/FIX) binding site of tissue factor could achieve similar clinical benefits in an arterial thrombosis model induced by surgical endarterectomy in chimpanzees. In this model, sequential surgical endarterectomies on right and left superficial femoral arteries were performed 30 days apart in five chimpanzees. A bolus (1 mg/kg) of ALT-836 was injected intravenously immediately preceding the restoration of flow in the endarterectomised femoral artery. Pre-surgical labelling of autologous platelets using (111)In-Oxine and post-surgical gamma camera imaging of (111)In-platelet deposition at endarterectomy sites was performed. The manipulated arterial segments were harvested for patency analysis 30 days following surgery. The results indicate that ALT-836 was highly effective at reducing acute vascular thrombosis, with no significant variations in surgical blood loss and template-bleeding time in the treated group compared to the control animals. These data suggest that ALT-836 is an effective and safe antithrombotic agent in preventing TF-initiated vascular thrombogenesis without compromising haemostasis.

BACKGROUND

Endothelial progenitor cells (EPC) are good candidates for cell-based therapy in cardiovascular diseases. However, concerns have been raised about the potential risks of EPC-based cell therapy, in terms of thrombogenicity particularly in inflammatory conditions, currently observed in such patients. Tissue factor (TF) can trigger coagulation and may support thrombogenicity. TF is also a key receptor in angiogenesis.

OBJECTIVE

The present study was designed to (i) evaluate the capacity of resting and tumour necrosis factor-alpha (TNF)-α-stimulated late-outgrowth endothelial colony-forming cells (ECFCs) to express TF and (ii) investigate the effect of TF/FVII(a) interaction on procoagulant and non-procoagulant activities of ECFCs in vitro.

METHODS

ECFCs from cord blood (cb) and adult peripheral blood (ab) were analyzed for TF expression and activity using reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, Western blot and a thrombin generation assay. Non-procoagulant properties of TF-expressing ECFCs were investigated in vitro using wound-healing, cell proliferation, tube formation and spheroid-based assays.

RESULTS

ECFCs expressed TF in response to TNF-α. The up-regulation of TF conferred to ECFCs a FVII(a)-dependent thrombin generation activity. Compared with cb-ECFC, ab-ECFCs can display a higher level of constitutive TF expression and activity, with a notable heterogeneity among donors. TF/FVIIa interaction did not modify non-procoagulant properties of TNF-α stimulated cb-ECFCs in vitro.

CONCLUSIONS

Proinflammatory conditions up-regulate TF expression in ECFCs. This expression confers to ECFCs a strong thrombin generation capacity without influencing their non-coagulant properties. Our results suggest that EPC-based cell therapy may be associated with prothrombotic risk which could be limited by inhibiting TF without affecting the proangiogenic capacity of the cells.

Oral direct inhibitors of thrombin and activated factor Xa are approved as new anticoagulant drugs. In contrast to vitamin K antagonists (VKA) and heparins, the new agents have single targets in the coagulation cascade and more predictable pharmacokinetics, but they lack validated and available antidotes. Unlike VKA, they do not require routine monitoring of coagulation. However, the measurement of their pharmacologic effects might be of value in selected patients. They interfere with the routine coagulation tests, which should be interpreted with caution. Specific tests exist and can be used in case of emergencies. Adequate supportive care and temporary removal of all antithrombotic agents constitute the basis for management of serious bleeding complications. The administration of coagulation factors, such as fresh frozen plasma, prothrombin complex concentrates or recombinant activated FVII, can benefit in life-threatening bleeding or emergency surgery. Specific antidotes for non-vitamin K oral anticoagulants are in clinical development. This review aims at answering in a brief and simplified manner some clinical questions.

Rare coagulation disorders (RCDs) include the inherited deficiencies of fibrinogen, factor (F) II, FV, combined FV and VIII, FVII, FX, combined FVII and X, FXI, FXIII and combined congenital deficiency of vitamin K-dependent factors (VKCFDs). Despite their rarity, a deep comprehension of all these disorders is essential to really understand haemostasis. Indeed, even if they share some common features each RCD has some particularity which makes it unique. In this review, we focus on three disorders: fibrinogen, FVII and FXIII.

Factor VII (FVII) activating protease (FSAP) is a circulating protease with a putative function in blood coagulation and fibrinolysis. Genetic epidemiological studies have implied a role for FSAP in carotid stenosis, stroke and thrombosis. To date, no in vivo evidence is available to support these claims. We have, for the first time, used FSAP-/- mice to define its role in thrombosis and haemostasis in vivo and to characterise the molecular mechanisms involved. FeCl3-induced arterial thrombosis in carotid and mesenteric artery revealed that the occlusion time was significantly increased in FSAP-/- mice (p< 0.01) and that some FSAP-/- mice did not occlude at all. FSAP-/- mice were protected from lethal pulmonary thromboembolism induced by collagen/ epinephrine infusion (p< 0.01). Although no spontaneous bleeding was evident, in the tail bleeding assay a re-bleeding pattern was observed in FSAP-/- mice. To explain these observations at a mechanistic level we then determined how haemostasis factors and putative FSAP substrates were altered in FSAP-/- mice. Tissue factor pathway inhibitor (TFPI) levels were higher in FSAP-/- mice compared to WT mice whereas FVIIa levels were unchanged. Other coagulation factors as well as markers of platelet activation and function revealed no significant differences between WT and FSAP-/- mice. This phenotype of FSAP-/- mice could be reversed by application of exogenous FSAP. In conclusion, a lack of endogenous FSAP impaired the formation of stable, occlusive thrombi in mice. The underlying in vivo effect of FSAP is more likely to be related to the modulation of TFPI rather than FVIIa.

A comparative study was performed on strong cation-exchangers to investigate the pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and SEM pictures of chromatographic resins. The resins tested included: SP Sepharose XL, Poros 50 HS, Toyopearl SP 550c, SP Sepharose BB, Source 30S, TSKGel SP-5PW-HR20, and Toyopearl SP 650c. Testing was performed with four different proteins: anti-FVII Mab (IgG), aprotinin, lysozyme, and myoglobin. Dependence of pH on retention was generally very low for proteins with high pI. An unexpected binding at pH 7.5 of anti-FVII Mab with pI < 7.5 was observed on several resins. Efficiency results show the expected trend of higher dependence of the plate height with increasing flow rate of soft resins compared to resins for medium and high-pressure operation. Determination of particle size distribution by two independent methods, Coulter counting and SEM, was in very good agreement. The mono-dispersed nature of Source 30S was confirmed. Binding to cation-exchange resins as a function of ionic strength varies depending on the specific protein. Generally, binding and elution at high salt concentration may be performed with Toyopearl SP 550c and Poros 50 HS, while binding and elution at low salt concentration may be performed with Toyopearl SP 650c. A very high binding capacity was obtained with SP Sepharose XL. Comparison of static capacity and dynamic capacity at 10% break-through shows in general approximately 50-80% utilisation of the total available capacity during chromatographic operation. A general good agreement was obtained between this study and data obtained by others. The results of this study may be used for selection of resins for testing in process development. The validity of experiments and results with model proteins were tested using human insulin precursor in pure state and in real feed-stock on Toyopearl SP 550c, SP Sepharose BB, and Toyopearl SP 650c. Results showed good agreement with experiments with model proteins.

It is widely recognized that thrombosis is the major event in the evolution of acute myocardial infarction (AMI) and acute ischemic stroke (AIS). But the contribution of coagulation factors to the development of ischemic arterial diseases is still not clearly established. The goal of this study was to establish the possible relationship between coagulation factors as well as anticoagulant and the onset of AMI and AIS. The study population consisted of 69 patients with AMI and 71 with AIS as well as 50 age-matched healthy volunteers. Compared with the control group, plasma tissue factor (TF) and tissue factor pathway inhibitor (TFPI) activities and both TF and TFPI antigens were significantly higher in the AMI group; plasma TF activity and antigen in AIS group were significantly increased, but the activity and antigen of plasma TFPI were significantly decreased in the AIS group. Plasma FVII coagulation (FVII:C) activity was markedly higher in patients with AIS, but not statistically different to the control in patients with AMI. FVIII coagulation (FVIII:C) activity was remarkably higher in patients with AMI but slightly lower than the control in patients with AIS. In the AMI and AIS groups, prothrombin activity and clottable fibrinogen were significantly higher and plasma antithrombin III activity was remarkably lower than the control. The results suggested that during the onset of AMI and AIS, the initiation of TF pathway would be associated with the thrombotic events and that the blood be in hypercoagulable state. But the changes of FVII:C, TFPI and FVIII:C in AMI are different from those in AIS.

Alterations in haemostasis are frequently observed in children with acute lymphoblastic leukemia (ALL). It was the objective of this study to analyse age-related disturbances in coagulation and fibrinolysis parameters during the induction phase of the antileukemic treatment. Sixty-four children were classified by age into three groups (1-5, 6-10, 11-16 years), and studied during induction treatment of ALL including four weeks of dexamethasone, followed by two weeks tapering of dexamethasone during which 6,000 IU/m(2) native L-Asparaginase (total 4 doses) was administered intravenously twice weekly. Blood samples were collected immediately before each L-Asparaginase infusion to analyze procoagulant (fibrinogen, factor [F] II, FV, FVII, F IX, F X) and anticoagulant factors (antithrombin [AT], protein C, protein S), parameters of thrombin generation (F1+2, TAT) and fibrinolysis (alpha2-antiplasmin, plasminogen, PAP, D-dimer). Children were in a hypercoagulable state after four weeks of dexamethasone due to upregulation of coagulation parameters. Upregulation was highest in the two youngest age groups. During L-Asparaginase treatment the 11- to 16-year-olds showed lower values in procoagulant and, even more, in anticoagulant factor levels compared to the younger children. Activation markers of thrombin generation and fibrinolysis did not change over time during the study period. Decreased synthesis of alpha2-antiplasmin and plasminogen during L-Asparaginase treatment resulted in less potential of clot lysis by plasmin in children older than 11 years of age. In conclusion, a more severe decline of anticoagulant and fibrinolytic parameters in children between 11 and 16 years of age underline that these children are at higher risk of thrombosis during ALL induction treatment.

Patients with haematological malignancies carry increased risk of venous thrombosis (VT). However, the mechanisms that link these malignancies to activated coagulation have not been fully identified. Since anti-haemostatic agents are studied in clinical trials for their potential to prolong survival in cancer patients, a detailed characterisation of haemostatic markers in cancer subtypes is needed. Hence, in this study, we measured the plasma concentrations and mRNA expression in blood mononuclear cells of haemostatic parameters in 93 patients with haematological neoplasias (acute myeloid leukaemia, chronic lymphatic leukaemia, multiple myeloma, and non-Hodgkin's lymphoma) before start and after completion of cancer therapy. At diagnosis we found activation of coagulation and fibrinolysis, especially in patients with acute myeloid leukaemia. This hypercoagulation was not associated with increased levels of tissue factor (TF) or factor VII (fVII) antigen or mRNA, or levels of activated fVII. In conclusion we found a hypercoagulable state in patients with haematological malignancy that did not seem to be initiated by TF.

Coagulation factor VIIa (FVIIa) is present at subnanomolar concentration and represents a small percentage of the total amount of FVII in the circulation. FVIIa is poised to initiate blood clotting when it encounters its pivotal cofactor tissue factor (TF) which becomes exposed to blood upon vascular rupture. The requirement for complex formation with TF in order for FVIIa to express procoagulant activity ensures thrombin and fibrin generation at the right time and place. Thus TF acts as a guardian of safety of paramount importance to blood coagulation by providing localization to the site of injury and at the same time inducing maturation of zymogen-like free FVIIa to the active cofactor-bound enzyme. This review gives an account of the accumulated knowledge about the structure, function and TF dependence of FVIIa to arrive at a plausible allosteric mechanism by which TF induces maturation of the active conformation of FVIIa.

Rare bleeding disorders (RBDs) are inherited deficiencies of coagulation factors such as fibrinogen, factor (F) II, FV, FVII, combined FV+FVIII, FX, FXI and FXIII. These disorders usually have a low prevalence in the general population and constitute approximately 3-5% of all coagulation disorders. However, in some countries they may have the same prevalence as haemophilia B due to the practice of consanguineous marriage. The clinical picture of RBDs is highly variable and can vary markedly from mild to severe, making both diagnosis and optimal treatment quite challenging. This review focuses on: (i) the efforts to establish a bleeding assessment tool adequate to RBDs, (ii) the optimal management of patients affected with FXI deficiency and (iii) the correlation between clinical severity and laboratory diagnosis when determining the minimum coagulant activity required to prevent bleeding in each RBD.

BACKGROUND

Hereditary factor VII (FVII) deficiency is characterized as a mild bleeding disorder in Beagles, caused by a missense mutation in exon 5 of the FVII gene. An Alaskan Klee Kai dog with severe bleeding after trauma was diagnosed with FVII deficiency based on coagulation testing. Molecular analyses were undertaken to identify the genetic basis of the defect in this breed.

OBJECTIVE

FVII deficiency in Alaskan Klee Kai dogs is caused by a mutation in the FVII gene.

METHODS

Eighteen client-owned Alaskan Klee Kai.

METHODS

Coagulation screening tests and factor assays were performed to characterize the coagulopathy. All coding regions of the propositus' FVII gene were sequenced. Amplification of exon 5, sequencing, and Mnl I restriction digest experiments were performed to screen for a point mutation in the remaining 17 dogs.

RESULTS

FVII deficiency was diagnosed in 6 dogs with a median FVII activity (FVII: C) of 5% (reference range, 50 150%). All FVII-deficient Alaskan Klee Kai were homozygous for the same mutation as FVII-deficient Beagles (ie, a G to A transition), resulting in substitution of glycine 96 by glutamic acid. An overlap in the FVII: C values obtained from heterozygote and wild-type dogs precluded accurate detection of carriers without genetic screening.

CONCLUSIONS

FVII deficiency may be associated with a bleeding tendency and should be considered in Alaskan Klee Kai dogs with prolonged prothrombin times. Plasma FVII: C accurately identifies affected dogs, but deoxyribonucleic acid testing is required for identification of carriers.

The FVII level is considered a risk factor for cardiovascular disease. Some of the polymorphic differences in the promoter of the F7 gene have been associated with variations in FVII levels. However, linkage disequilibrium among those polymorphisms has made it difficult to pinpoint the true functional variants, so contradictory results have often appeared among various studies. We provide new findings of the effect of the polymorphisms in the promoter region of F7. In vitro transfection of 15 plasmids containing different combinations of F7 promoter polymorphisms was performed in HepG2 cells. We found that allelic variants -323ins10 and -122C strongly reduced promoter activity and that allelic variant -402A significantly increased promoter activity. We report the effect of a novel variant (-2989A) that significantly increases F7 expression levels. However, this novel allelic variant is in strong linkage disequilibrium with the -323ins10 variant in our Spanish population, which has a clear dominant effect over the -2989A variant and completely masks its effect. Our results have important implications for mapping genes affecting complex diseases using association studies. That is, they imply that true functional variants should be chosen to confirm the analyses and to ensure that the results can be reproduced in other populations. In addition, our results suggest that it would be informative to screen for the -2989A variant in other populations, since it may well be a risk factor for cardiovascular disease in populations where it does not appear with the decanucleotide insertion.

Recombinant activated factor VII (rFVIIa) is an hemostatic agent that was originally developed for the treatment of hemorrhage in patients with hemophilia and inhibitors. However, in the last few years rFVIIa has been employed with success in a broad spectrum of congenital and acquired bleeding conditions. In this systematic review we present the current knowledge on the use of this drug in patients suffering from hemato-oncological disorders, which are quite commonly complicated by severe hemorrhage. On the whole, data in the literature suggest a potential role for rFVIIa in the management of bleeding unresponsive to standard therapy in patients with hematological malignancies, including those undergoing bone marrow transplant. However, the vast majority of the currently available data are derived from uncontrolled studies including single cases or small series of patients. Thus, further trials with larger numbers of patients are needed to establish the most appropriate doses and timing of rFVIIa and to assess its efficacy and safety in this setting.

We investigated the effects of low lipoprotein receptor deficiency in cholesterol blood concentrations, blood pressure, hemostatic factors, and the autonomic nervous system in three groups: control mice fed standard diet (CO, n=9), lipoprotein receptor-deficient mice (LDLr(-/-), n=9) fed standard diet (LDLr-S) or hypercholesterolemic diet (LDLr-H, n=8). Frequency domain analysis of heart rate and blood pressure variability was performed with an autoregressive algorithm. The spectral components were expressed in absolute (s(2) or mmHg(2)) and normalized units. Spontaneous baroreflex sensitivity (BRS) was estimated by alpha index, defined as square root ratio between low frequency power in blood pressure variability and heart rate variability. LDLr/- mice presented a significant increase in the cholesterol blood concentration (mean±SD; mg/dl; LDLr-S=202.01±34.38 and LDLr-H=530.7±75.17) compared to CO (79.2±13.6), p=0.001. The receptor deletion was associated with a heart rate variability reduction (p=0.013). The BRS was reduced (p<0.05) in LDLr-S and LDL-H (mean±SD: 0.96±0.39 and 0.59±0.34, respectively) compared to CO (4.02±1.92). Moreover, hypercholesterolemic diet significantly increased the cardiac sympathetic modulation (0V pattern of symbolic analysis: mean±SD, CO=8.04±4.53; LDLr-S=16.49±4.52 and LDLr-H=21.80±8.24, p=0.006). The 0V pattern was statically correlated to coagulation factor VII (r=0.555, p=0.0208). In LDLr-H, the concentration (interquartile range) of plasmatic fibrinogen and hemostatic factors VII (2.8-3.3) and XII (1.1-1.3) were increased compared to CO (0.9-1.1and 0.9-1.0, respectively) and LDLr-S (0.7-1.0 and 0.8-0.9, respectively) (p<0.004 for FVII and p<0.006 for FXII). Taken together, the results indicate that plasmatic cholesterol magnitude is determinant to increase the coagulation and the sympathetic modulation.

Severe inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder with a poor relationship between FVII coagulant activity and bleeding tendency. Both clinical expression and mutational spectrum are highly variable. We have screened for mutations the FVII gene of 37 unrelated patients with a FVII coagulant activity less than 5% of normal pooled plasmas. The nine exons with boundaries and the 5' flanking region of the FVII gene were explored using a combination of denaturing gradient gel electrophoresis and direct DNA sequencing. This strategy allowed us to characterise 68 out of the 74 predicted FVII mutated alleles. They corresponded to a large panel of 40 different mutations. Among these, 18 were not already reported. Genotypes of the severely affected patients comprised, on both alleles, deleterious mutations which appeared to be related to a total absence of activated FVII. We suggest that this absence of functional FVII can explain the severe clinical expression. Whether a small release of FVII is sufficient to initiate the coagulation cascade and to prevent the expression of a severe phenotype, requires further investigations.

BACKGROUND

Chronic obstructive pulmonary disease (COPD) is associated with an increased risk for thromboembolic events. We investigated thrombin generation profiles in COPD patients and their dependence on plasma factor/inhibitor composition.

METHODS

Factors (f) (fII, fV, fVII, fVIII, fIX, fX), antithrombin, protein C (PC) and free tissue factor pathway inhibitor (fTFPI) from 60 COPD patients (aged 64.2 ± 10.1 years; a mean forced expiratory volume in 1 second [FEV(1)], 55.6 ± 15.8% of predicted values) were compared with those for 43 controls matched for age, sex, weight and smoking. Patients receiving anticoagulation were excluded. Using each individual's plasma coagulation protein composition, tissue factor-initiated thrombin generation was assessed computationally.

RESULTS

COPD patients had higher fII (115 ± 16 vs 102 ± 10%, p < 0.0001), fV (114 ± 19 vs 102 ± 12%, p = 0.0002), fVII (111 ± 15 vs 102 ± 17%, p = 0.002), fVIII (170 ± 34 vs 115 ± 27%, p < 0.0001), and fIX (119 ± 21 vs 107 ± 17%, p = 0.003), and lower fTFPI (17.7 ± 3.2 vs 18.9 ± 3.2 ng/ml, p = 0.047) compared with controls, while fX, antithrombin, and PC were similar in both groups. Computational thrombin generation profiles showed that compared with controls, COPD patients had higher maximum thrombin levels (+28.3%, p < 0.0001), rates of thrombin generation (+46.1%, p < 0.0001) and total thrombin formation (+14.4%, p < 0.001), together with shorter initiation phase of thrombin generation (p < 0.0001) and the time to maximum thrombin levels (p < 0.0001). Thrombin generation profiles in COPD patients can be normalized via correction of fII, fVIII , fIX and TFPI. The severity of COPD and inflammatory markers were not associated with thrombin generation profiles.

CONCLUSIONS

Prothrombotic phenotype in COPD patients is largely driven by increased prothrombin, fVIII, fIX, and lower fTFPI.

The use of recombinant FVIIa (rFVIIa) to control bleed in individuals with FVII deficiency has been proven to be effective. The main problems associated with its use are that it requires frequent bolus injections to counteract its short half-life and high cost. Our study aimed to evaluate whether any advantage could be gained by providing rFVIIa by continuous infusion during surgery with regard to haemostatic efficacy, safety and cost. The prospective study included 10 patients with severe FVII deficiency, who underwent 25 surgical procedures (13 major and 12 minor procedures) and were treated with rFVIIa administered by continuous infusion. Tranexamic acid was given concomitantly every 8 h. Prothrombin time, FVII:C assay and thrombin generation assay were used to monitor the treatment. The mean total dose given was 10 mg during a major surgery and 4.4 mg during a minor surgery for a mean treatment duration of 7.5 and 4.0 days respectively. This corresponds to a reduction of 70-90% in drug usage and medication cost compared with bolus injections. Except for one major perioperative bleeding, excellent haemostasis was achieved in all procedures. One patient developed a transient inhibitory activity. None of these events affected the postoperative course or prolonged the hospital stay. Our study demonstrated that continuous infusion of rFVIIa during surgery is safe, effective and highly cost effective.

There is evidence that high plasma levels of factor (F) VIII, FIX, FXI and fibrinogen are independent risk factors for venous thromboembolism.

OBJECTIVE

To determine the plasma concentrations of several coagulation factors and C4b-binding protein (C4BP) in a group of patients with non-metastatic colorectal cancer in order to investigate some aspects of cancer-acquired thrombophilia.

METHODS

Plasma fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI and FXII activity levels and C4BP concentrations were determined in 73 patients with non-metastatic colorectal cancer (48 colon and 25 rectum) and in 67 matched control subjects. No one in either group had had previous thrombotic events.

RESULTS

Mean plasma concentrations of fibrinogen (functional and antigen), FVIII, FIX, FV and C4BP were significantly higher in colorectal cancer patients than in control subjects, while FVII and FXII levels were significantly decreased. Several correlations were found between the increased coagulation factors and C4BP concentrations, while FVII was highly correlated with FXII.

CONCLUSIONS

In colorectal cancer patients high plasma fibrinogen, FVIII and FIX levels might represent further risk factors for venous thrombotic complications in the immediate post-surgery period, while decreased FVII and FXII concentrations may be an index of intravascular coagulation activation, still in a subclinical phase.

BACKGROUND

FFP is considered adequate for transfusion up to 24 hours after thawing and is currently used most often to replace deficient clotting factors, such as in warfarin overdose. We set to examine the levels of vitamin K-dependent factors (i.e., prothrombin, FVII, F IX, FX), as well as fibrinogen, upon twice freezing and thawing of FFP. If factor levels in refrozen FFP remain within normal limits, this component can possibly be transfused, thus avoiding wastage of precious blood components.

METHODS

Twenty units of FFP, five units of each blood group A, B, AB, and O, were thawed, and aliquots were taken for measurement of coagulation factors. The plasma units were then kept for 24 hours at 4 degrees C, at which point a second aliquot was taken, The remaining FFP units were refrozen and kept at -80 degrees C for 1 week. The above procedure was then repeated. Coagulation-factor activity and fibrinogen level were measured by the coagulation analyzer.

RESULTS

The mean levels of prothrombin, FVII, F IX, FX, and fibrinogen of each blood group (A, B, AB, and O) were calculated for each of four time points and found not statistically different (p>> 0.05). Therefore, the rest of the analysis was done for all 20 FFP units as one group. The mean +/- SD levels of each coagulation factor at each time point demonstrated that all levels were within normal limits of all factors measured and that for none of the factors was there a significant decay of activity.

CONCLUSIONS

The levels of prothrombin, FVII, F IX, FX, and fibrinogen remain stable and adequate for transfusion in twice-thawed-and-refrozen FFP. This component can be safely used for transfusion as a source of vitamin K-dependent clotting factors and fibrinogen.

Paradoxically, there are reports of thrombotic events for some rare bleeding disorders associated with significant bleeding tendency. Afibrinogenemia, factor (F) VII, or FXI deficiencies are those most commonly associated with venous or arterial thrombosis. Pathogenesis is multifactorial and the main conditions associated with this complication relate to the coexistence of inherited or acquired thrombotic risk factors linked to certain specific characteristics of the underlying defect. Patients with afibrinogenemia can develop severe, spontaneous, or recurrent thromboembolic disease. Up to 20% of congenital dysfibrinogenemia patients show predisposition to thrombosis. Thrombotic episodes, particularly deep vein thrombosis, have been reported in 3 to 4% FVII deficient patients, even those who were severely affected. These events have been reported either after infusion of plasma derived FXI concentrate or recombinant activated FVII in FXI deficient patients. So, in addition to factor level, replacement therapy must be individualized and should take into account past personal or family history of bleeding and thrombosis, and other prothrombotic risk factors. Treatment of thrombosis represents a challenge. For mild factor deficiencies, antithrombotic prophylaxis must be considered with or without concomitant use of replacement therapy. For all patients, it is also recommended to control known cardiovascular disease risk factors.

OBJECTIVE

An increase of FVII activity (FVIIc) has been proposed to be an independent cardiovascular risk factor. Whether FVII is associated with insulin resistance in coronary heart disease (CHD) is still unknown. We tested the hypothesis that plasma FVII activity and leptin are associated with insulin resistance independently.

METHODS

We studied 130 subjects, of which 65 were CHD subjects and 65 were non-CHD control subjects. Fasting plasma levels of leptin, insulin, glucose, FVIIc activity, fibrinogen, lipid parameters were estimated for all the subjects. Body mass index (BMI), waist circumference (WC) and blood pressure levels were also determined.

RESULTS

We observed significantly raised plasma levels of FVIIc activity, leptin and insulin resistance among the CHD subjects compared to the non-CHD subjects. Raised FVIIc activity levels in CHD were significantly positively correlated with insulin resistance. Raised plasma leptin levels in CHD were correlated with insulin resistance, BMI and WC. Multivariate regression analysis showed that elevated levels of FVII activity in CHD was significantly associated with insulin resistance, independent of dyslipidemia, leptin, blood pressure levels, BMI, WC, gender and age. Furthermore, raised leptin levels in CHD subjects were significantly associated with insulin resistance and BMI, independently of each other and of dyslipidemia, FVIIc, blood pressure levels, WC, gender and age.

CONCLUSIONS

Raised FVII and leptin levels in CHD patients were independently associated with insulin resistance, this was not observed among the non-CHD subjects.

Blood coagulation activation might be one mechanism linking acute mental stress with coronary events. We investigated the natural habituation of coagulation responses and recovery to short-term mental stress. Three times with one-week intervals, 24 men (mean age 47 +/- 7 years) underwent the same 13-min stressor (preparation, job interview, mental arithmetic). During each visit venous blood was obtained four times (baseline, immediately post-stress, 45 min of recovery, 105 min of recovery). Eight blood coagulation parameters were measured at weeks one and three. Acute stress provoked increases in von Willebrand factor antigen, fibrinogen, clotting factor FVII activity (FVII:C), FVIII:C, FXII:C (p's < or = 0.019), and D-dimer (N.S.). All coagulation parameters experienced full recovery except FVIII:C (p = 0.022). Stress did not significantly affect activated partial thromboplastin time and prothrombin time. At all time points FVIII:C and FXII:C levels were significantly higher at week one compared to week three (p's < or = 0.041). Before catheter insertion, systolic blood pressure (p = 0.001) and heart rate (p = 0.026) were relatively higher at week one. Unlike the magnitude of systolic blood pressure response to stress (p = 0.007) and of cortisol recovery from stress (p = 0.002), the magnitude of all coagulation responses to stress and the recovery from stress were similar in week one and week three. Sympathetic activation with anticipatory stress best explained increased baseline activity in FVIII and FXII at week one. An incapacity of the coagulation system to adapt to stress repeats is perhaps a consequence of evolution, but might also contribute to increased coronary risk in some individuals, particularly in those with cardiovascular diseases.