Intrinsic Coagulation Pathway, History of Headache, and Risk of Ischemic Stroke.

Background and Purpose- Hypercoagulable states in migraine patients may play a role in the pathophysiology underlying the association between migraine and ischemic stroke. This study aims to provide more insight into the potential association of headache, ischemic stroke, and the intrinsic coagulation pathway. Methods- We included patients from the RATIO study (Risk of Arterial Thrombosis in Relation to Oral Contraceptives), a Dutch population-based case-control study including young women (age <50) with ischemic stroke and healthy controls. We defined a headache group based on a questionnaire on headache history. Intrinsic coagulation proteins were measured through both antigen levels (FXII, FXI, prekallikrein, HK [high molecular weight kininogen]) and protein activation, determined by measuring activated protein complex with C1esterase-inhibitor (FXIIa-C1-INH, FXIa-C1-INH, Kallikrein-C1-INH) or antitrypsin-inhibitor (FXIa-AT-INH). We calculated adjusted odds ratios and performed an interaction analysis assessing the increase in stroke risk associated with high levels of intrinsic coagulation and history of headache. Results- We included 113 ischemic stroke cases and 598 healthy controls. In total, 134 (19%) patients had a history of headache, of whom 38 were cases and 96 controls. The combination of headache and high intrinsic coagulation protein levels (all but FXII antigen level and both FXIa-inhibitors) was associated with an increase in ischemic stroke risk higher than was expected based on their individual effects (adjusted odds ratio FXI antigen level alone: 1.7, 95% CI, 1.0-2.9; adjusted odds ratio headache alone: 2.0, 95% CI, 1.1-3.7; combination: 5.2, 95% CI, 2.3-11.6) Conclusions- Headache and high intrinsic coagulation protein levels may biologically interact, increasing risk for ischemic stroke.

Factor XIIa regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin.

Recent data indicate an important contribution of coagulation factor (F)XII to in vivo thrombus formation. Because fibrin structure plays a key role in clot stability and thrombosis, we hypothesized that FXII(a) interacts with fibrin(ogen) and thereby regulates clot structure and function. In plasma and purified system, we observed a dose-dependent increase in fibrin fiber density and decrease in turbidity, reflecting a denser structure, and a nonlinear increase in clot stiffness with FXIIa. In plasma, this increase was partly independent of thrombin generation, as shown in clots made in prothrombin-deficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and α-FXIIa, but not ß-FXIIa, bound to purified fibrinogen and fibrin with nanomolar affinity. Immunostaining of human carotid artery thrombi showed that FXII colocalized with areas of dense fibrin deposition, providing evidence for the in vivo modulation of fibrin structure by FXIIa. These data demonstrate that FXIIa modulates fibrin clot structure independently of thrombin generation through direct binding of the N-terminus of FXIIa to fibrin(ogen). Modification of fibrin structure by FXIIa represents a novel physiologic role for the contact pathway that may contribute to the pathophysiology of thrombosis.

Early atherosclerosis exhibits an enhanced procoagulant state.

BACKGROUND: Thrombin generation in vivo may be important in regulating atherosclerotic progression. In the present study, we examined for the first time the activity and presence of relevant coagulation proteins in relation to the progression of atherosclerosis. METHODS AND RESULTS: Both early and stable advanced atherosclerotic lesions were collected pairwise from each individual (n=27) during autopsy. Tissue homogenates were prepared from both total plaques and isolated plaque layers, in which the activity of factors (F) II, X, and XII and tissue factor was determined. Microarray analysis was implemented to elucidate local messenger RNA synthesis of coagulation proteins. Part of each specimen was paraffin embedded, and histological sections were immunohistochemically stained for multiple coagulation markers with the use of commercial antibodies. Data are expressed as median (interquartile range [IQR]). Tissue factor, FII, FX, and FXII activities were significantly higher in early atherosclerotic lesions than in stable advanced atherosclerotic lesions. Endogenous thrombin potential and thrombin-antithrombin complex values consolidated a procoagulant profile of early atherosclerotic lesions (endogenous thrombin potential, 1240 nmol/L x min [IQR, 1173 to 1311]; thrombin-antithrombin complex, 1045 ng/mg [IQR, 842.6 to 1376]) versus stable advanced atherosclerotic lesions (endogenous thrombin potential, 782 nmol/L x min [IQR, 0 to 1151]; thrombin-antithrombin complex, 718.4 ng/mg [IQR, 508.6 to 1151]). Tissue factor, FVII, and FX colocalized with macrophages and smooth muscle cells. In addition, multiple procoagulant and anticoagulant proteases were immunohistochemically mapped to various locations throughout the atherosclerotic vessel wall in both early and advanced atherosclerotic stages. CONCLUSIONS: This study shows an enhanced procoagulant state of early-stage atherosclerotic plaques compared with advanced-stage plaques, which may provide novel insights into the role of coagulation during atherosclerotic plaque progression.

Misfolded proteins activate factor XII in humans, leading to kallikrein formation without initiating coagulation.

When blood is exposed to negatively charged surface materials such as glass, an enzymatic cascade known as the contact system becomes activated. This cascade is initiated by autoactivation of Factor XII and leads to both coagulation (via Factor XI) and an inflammatory response (via the kallikrein-kinin system). However, while Factor XII is important for coagulation in vitro, it is not important for physiological hemostasis, so the physiological role of the contact system remains elusive. Using patient blood samples and isolated proteins, we identified a novel class of Factor XII activators. Factor XII was activated by misfolded protein aggregates that formed by denaturation or by surface adsorption, which specifically led to the activation of the kallikrein-kinin system without inducing coagulation. Consistent with this, we found that Factor XII, but not Factor XI, was activated and kallikrein was formed in blood from patients with systemic amyloidosis, a disease marked by the accumulation and deposition of misfolded plasma proteins. These results show that the kallikrein-kinin system can be activated by Factor XII, in a process separate from the coagulation cascade, and point to a protective role for Factor XII following activation by misfolded protein aggregates.

Dual role of collagen in factor XII-dependent thrombus formation.

In vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets via glycoprotein VI activation. Furthermore, in flow-dependent thrombus formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the formation of fibrin. Defective glycoprotein VI signaling (with platelets deficient in LAT or phospholipase Cgamma2) delayed and suppressed phosphatidylserine exposure and thrombus formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factor VIIa was of little effect. Together, these results point to a dual role of collagen in thrombus formation: stimulation of glycoprotein VI signaling via LAT and PLCgamma2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi.

Endothelial activation in lacunar stroke subtypes.

BACKGROUND AND PURPOSE: Lacunar stroke (LS) can be subtyped according to the absence (isolated lacunar infarct [ILA]) or presence of concomitant white matter lesions (WML) and/or asymptomatic lacunar infarcts. Endothelial activation is thought to play a pivotal role in the subtype with WML and/or asymptomatic lacunar infarcts. The aim of this study was to evaluate whether endothelial activation is associated with WML and/or asymptomatic lacunar infarcts in LS patients. Here, we determined levels of circulating blood markers of endothelial function in LS patients. METHODS: In 149 patients, all of whom had brain-MRI, levels of tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), tPA-PAI-1 complex, von Willebrand factor, tissue factor, thrombomodulin, and coagulation factor VIII were determined. Levels of blood markers were related to subtypes of LS and adjusted for age, gender, and vascular risk factors. RESULTS: In subtypes of LS, tPA activity was increased in patients with WML (0.79 IU/mL vs 0.44 IU/mL for ILA; P=0.02) and PAI-1-antigen levels were lowest in patients with WML (27.5 ng/mL vs 44.0 ng/mL for ILA; P=0.02). The association between WML and PAI-1 remained significant after multivariable analysis (OR, 0.99; 95% CI, 0.98-1.00 per ng/mL change of PAI-1; P=0.04). CONCLUSIONS: We found further evidence for the hypothesis of endothelial activation in the subtype of LS caused by a diffuse small vessel vasculopathy, as we found higher levels of tPA in patients with concomitant extensive WML than in those with ILA. Second, low levels of PAI-1 were associated with WML. We postulate that differences in activity of components of the fibrinolytic system might contribute to WML development.

A history of late and very late stent thrombosis is not associated with increased activation of the contact system, a case control study.

BACKGROUND: The pathophysiological pathways resulting in Late Stent Thrombosis (LST) remain uncertain. Findings from animal studies indicate a role of the intrinsic coagulation pathway in arterial thrombus formation, while clinical studies support an association with ischemic cardiovascular disease. It is currently unknown whether differences in the state of the contact system might contribute to the risk of LST or Very Late Stent Thrombosis (VLST). We assessed the relation between levels of several components involved in the contact system and a history of LST and VLST, termed (V)LST in a cohort of 20 patients as compared to a matched control group treated with PCI. METHODS AND RESULTS: Activated factor XII (FXIIa), FXII zymogen (FXII), FXIIa-C1-esterase inhibitor (C1-inhibitor), Kallikrein-C1-inhibitor, FXIa-C1-inhibitor and FXIa-alpha1-antitrypsin (AT-inhibitor) complexes were measured by Enzyme-linked immunosorbent assy (ELISA) methodology.Cases and controls showed similar distributions in sex, age, baseline medications and stent type. Patients with a history of (V)LST had a significantly greater stent burden and a higher number of previous myocardial infarctions than the control patients.There were no significant between-group differences in the plasma levels of the components of the contact system. CONCLUSION: In a cohort of patients with a history of (V)LST, we did not observe differences in the activation state of the intrinsic coagulation system as compared to patients with a history of percutaneous coronary intervention without stent thrombosis.

Monitoring thrombin generation: is addition of corn trypsin inhibitor needed?

Thrombin generation monitoring has the potential to be used as a clinical diagnostic tool in the near future. However, robust preanalytical conditions may be required, and one factor that has been reported is in-vitro contact activation that might influence in-vitro measurements of thrombin generation and thereby act as an unpredictable pre-analytical variable. The aim of the current study was to investigate the influence of contact activation and the necessity of corn trypsin inhibitor (CTI) to abolish contact activation in thrombin generation measurements at low tissue factor (TF) concentrations. Thrombin generation was performed using the calibrated automated thrombinoscopy (CAT), thereby determining the endogenous thrombin potential (ETP), peak height, and the lag time, in plasma obtained from healthy volunteers. Addition of CTI after plasma preparation had no significant influence on thrombin generation triggered with 0.5 pM TF or higher, as demonstrated by unaltered ETP and lag time values between analyses with and without CTI. Addition of CTI before blood collection reduced thrombin generation triggered with 0.5 pM TF: both the ETP and peak height were significantly reduced compared to no CTI addition. In contrast, thrombin generation remained unaltered at a 1 pM TF trigger or above. This study demonstrates that addition of CTI after plasma separation is not necessary when triggering with TF concentrations of 0.5 pM and higher. Furthermore, it was demonstrated that it is not needed to pre-fill blood collecting tubes with CTI when measuring thrombin generation at TF concentrations of >/=1 pM.

Impact of Deficiency of Intrinsic Coagulation Factors XI and XII on Ex Vivo Thrombus Formation and Clot Lysis.

The contributions of coagulation factor XI (FXI) and FXII to human clot formation is not fully known. Patients with deficiency in FXI have a variable mild bleeding risk, whereas FXII deficiency is not associated with bleeding. These phenotypes make FXII and FXI attractive target proteins in anticoagulant therapy. Here, we studied the mechanisms of fibrin clot formation, stability, and fibrinolytic degradation in patients with severe FXI or FXII deficiency. Thrombin generation was triggered in platelet-poor (PPP) and platelet-rich plasma (PRP) with the biological FXII trigger sulfatides. Intrinsic and extrinsic thrombus formation and degradation in whole blood were determined with rotational thromboelastometry (ROTEM). Clot formation under flow was assessed by perfusion of whole blood over collagen microspots with(out) tissue factor (TF). Thrombin generation and clot formation were delayed in FXII- and FXI-deficient patients triggered with sulfatides. In FXI-deficient plasma, this delay was more pronounced in PRP compared to PPP. In whole blood of FXII-deficient patients, clots were smaller but resistance to fibrinolysis was normal. In whole blood of FXI-deficient patients, clot formation was normal but the time to complete fibrinolysis was prolonged. In flow chamber experiments triggered with collagen/TF, platelet coverage was reduced in severe compared with moderate FXI deficiency, and fibrin formation was impaired. We conclude that quantitative defects in FXII and FXI have a substantial impact on contact activation-triggered coagulation. Furthermore, FXI deficiency has a dose-dependent suppressing effect on flow-mediated and platelet/TF-dependent clot formation. These last data highlight the contribution of particularly FXI to hemostasis.

Discovery and assessment of water soluble coumarins as inhibitors of the coagulation contact pathway.

Over the last decade, the coagulation factor XIIa (FXIIa) has seen renewed interest as a therapeutic target. Indeed, its inhibition could offer a protection against thrombosis without increasing the risk of bleeding. Moreover, it could answer the need for a safe prevention of blood-contacting medical devices-related thrombosis. Among the FXII and FXIIa inhibitors already described in literature, organic small-molecular-weight inhibitors are rather left behind. In this study, we were focused on the discovery and assessment of water soluble small molecules. First, a search within our library of compounds flagged two promising hits. Indeed, enzymes and plasma assays suggested they have a greater activity on the contact factors (FXIa, plasma kallikrein and FXIIa) than on the TF pathway. Then, simple pharmacomodulations were undertaken with the aim to design more selective FXIIa inhibitors. This afforded compounds having different degrees of selectivity. All compounds were finally screened in whole blood using an 8-channel microfluidic model and thromboelastometry measurements. Interestingly, all molecules interfered with the thrombus formation and one of them could be considered as a small organic contact inhibitor.

Fibrin clot formation and fibrinolysis in patients with a history of coronary stent thrombosis.

INTRODUCTION: Coronary stent thrombosis is a devastating complication of percutaneous coronary intervention (PCI). Multiple factors underlie the pathophysiological mechanisms of stent thrombosis. Previous studies demonstrated that patients with stent thrombosis, compared to control PCI patients, formed denser fibrin clots in vitro which were more resistant to fibrinolysis, suggesting that altered fibrin clot properties may contribute to the pathophysiology of stent thrombosis. We assessed the plasma fibrin clot formation and fibrinolysis of patients with and without stent thrombosis. METHODS: Cases (patients with stent thrombosis) and matched controls (patients without stent thrombosis) were included for a matched case-control study. Matching was performed on indication and time of the index PCI (initial stent implantation) from the cases. Fibrin clot formation and fibrinolysis were assessed in vitro by turbidimetric assays, with human thrombin to initiate fibrin polymerization and tissue type plasminogen activator to initiate fibrinolysis. Lag time, maximal absorbance and clot lysis time were determined by these assays. RESULTS: In total, 27 cases and 27 controls were included. No significant differences were observed between cases and controls in lag time (173 vs. 162s, p=0.18), maximal absorbance (0.78 vs. 0.83, p=0.36), and clot lysis time (69 vs. 71min, p=0.78). Fibrin clot formation and fibrinolysis were not associated with stent thrombosis. CONCLUSIONS: Plasma fibrin clot formation and fibrinolysis were not significantly different between patients with stent thrombosis and matched control patients, suggesting that fibrin clot formation and fibrinolysis play no significant role in the pathophysiology of stent thrombosis.

The role of activated coagulation factor XII in overall clot stability and fibrinolysis.

Activated coagulation factor XII (α-FXIIa) is able to bind to fibrin(ogen) and increases the density and stiffness of the fibrin clot. Conversely, proteins of the contact system and the fibrinolytic system show a high degree of homology and α-FXIIa can convert plasminogen into plasmin resulting in fibrin degradation. Therefore, we studied the contribution of α-FXIIa to overall clot stability and plasmin driven fibrinolysis in the absence and presence of tissue plasminogen activator (tPA). We observed that α-FXIIa directly converted plasminogen into plasmin and reduced clot lysis time at all tPA concentrations tested (15-1500 pM). Simultaneous assessment of plasmin generation (chromogenic substrate S-2251) and fibrin formation and degradation (absorbance at 405nm), showed an earlier onset of fibrinolysis and plasmin formation in the presence of α-FXIIa. Fibrinolysis of clots formed under flow conditions, revealed that incorporation of α-FXIIa accelerated clot breakdown (fluorescence release of labeled fibrin) by additional plasmin generation on top of formation by tPA. Scanning electron microscopy (SEM) revealed that the surface area pore size increased in the presence compared with the absence of α-FXIIa when fibrinolysis was initiated by the conversion of plasminogen with tPA during clot formation. α-FXIIa enhances fibrinolysis in the presence of plasminogen, irrespective of whether tPA was present during clot formation or was added afterwards to initiate fibrinolysis. We postulate that FXIIa first strengthens the clot structure during clot formation and thereafter contributes towards fibrinolysis.

Hypoxia Induces a Prothrombotic State Independently of the Physical Activity.

Hypoxia (oxygen deprivation) is known to be associated with deep vein thrombosis and venous thromboembolism. We attempted to get a better comprehension of its mechanism by going to high altitude, thereby including the potential contributing role of physical activity. Two groups of 15 healthy individuals were exposed to hypoxia by going to an altitude of 3900 meters, either by climbing actively (active group) or transported passively by cable car (passive group). Both groups were tested for plasma fibrinogen, von Willebrand factor and factor VIII levels, fibrinolysis, thrombin generating capacity, heart rate, oxygen saturation levels and blood pressure. As a control for the passive group, 7 healthy volunteers stayed immobile in bed for 7 days at normoxic conditions. The heart rate increased and oxygen saturation levels decreased with increasing altitude. Fibrinolysis and fibrinogen levels were not affected. Factor VIII and von Willebrand factor levels levels increased significantly in the active group, but not in the passive group. Plasma thrombin generation remained unchanged in both the active and passive group with increasing altitude and during 7 days of immobility in healthy subjects. However, by applying whole blood thrombin generation, we found an increased peak height and endogenous thrombin potential, and a decreased lagtime and time-to-peak with increasing levels of hypoxia in both groups. In conclusion, by applying whole blood thrombin generation we demonstrated that hypoxia causes a prothrombotic state. As thrombin generation in plasma did not increase, our results suggest that the cellular part of the blood is involved in the prothrombotic phenotype induced by hypoxia.

Prothrombotic markers in familial combined hyperlipidemia: evidence of endothelial cell activation and relation to metabolic syndrome.

BACKGROUND: Familial combined hyperlipidemia (FCHL) is characterized by a varied expression of hypertriglyceridemia and hypercholesterolemia within a family, and a high risk of premature coronary artery disease. The present study evaluated a number of potential prothrombotic markers in familial combined hyperlipidemia, and studied their relationship to the hypercholesterolemic (Fredrickson type IIa) and hypertriglyceridemic (IIb and IV) phenotypes. METHODS AND RESULTS: Selected prothrombotic markers were studied in 68 subjects: 34 hyperlipidemic subjects with familial combined hyperlipidemia and 34 controls. FCHL patients exhibited significantly higher Thrombin-Antithrombin complex (TAT), activated coagulation factor XII (F XIIa), von Willebrand Factor (vWF), Plasminogen Activator Inhibitor-1 (PAI-1) and tissue derived Plasminogen Activator (t-PA) values in comparison to controls. Within the subgroup of familial combined hyperlipidemia subjects, elevated PAI-1 activity and soluble Thrombomodulin levels were particularly associated with features of the metabolic syndrome, including hyperinsulinemia, hypertriglyceridemia and predominance of small dense low density lipoprotein (LDL). CONCLUSIONS: A general pattern of activated blood coagulation and endothelial activation is present in all hyperlipidemic subjects studied, independent of metabolic phenotype. In those familial combined hyperlipidemia subjects with features of the metabolic syndrome, impaired fibrinolysis can provide an additional cardiovascular risk factor.

Reduced factor V concentration and altered FV1/FV2 ratio do not fully explain R2-associated APC-resistance.

Carriership of the factor V (FV) R2 haplotype is associated with mild APC-resistance, moderately reduced FV levels and a relative increase of the more thrombogenic FV isoform, FV1. Since low FV levels and increased FV1 can theoretically cause APC-resistance, we investigated whether these alterations can quantitatively account for R2-associated APC-resistance. In order to determine the effect of FV concentration and FV isoform composition on the APC-response, we reconstituted FV-deficient plasma with purified FV1 and FV2 in different molar ratios and to varying FV concentrations. APC sensitivity ratios (APCsr) were determined with the Coatest APC Resistance V, which probes the effect of APC on both FVa- and FVIIIa-inactivation, and with the Immunochrom APC response test, which only quantifies the effect of APC on FVIII(a)-inactivation. In both assays, low FV concentrations and/or high relative amounts of FV1 rendered plasma samples more resistant to APC. APCsr were also determined in FV-deficient plasma reconstituted with purified FV at levels and isoform ratios observed in R2-homozygotes (98% FV, 42% FV1) and age-matched controls (119% FV, 26% FV1). In both tests the APCsr of reconstituted control plasma was the same as that of plasma from controls, whereas reconstituted R2-plasma was less APC-resistant than plasma from homozygous carriers of the R2 haplotype. We conclude that the low FV levels and altered FV isoform ratio cannot fully explain R2-associated APC-resistance.

Ongoing contact activation in patients with hereditary angioedema.

Hereditary angioedema (HAE) is predominantly caused by a deficiency in C1 esterase inhibitor (C1INH) (HAE-C1INH). C1INH inhibits activated factor XII (FXIIa), activated factor XI (FXIa), and kallikrein. In HAE-C1INH patients the thrombotic risk is not increased even though activation of the contact system is poorly regulated. Therefore, we hypothesized that contact activation preferentially leads to kallikrein formation and less to activation of the coagulation cascade in HAE-C1INH patients. We measured the levels of C1INH in complex with activated contact factors in plasma samples of HAE-C1INH patients (N=30, 17 during remission and 13 during acute attack) and healthy controls (N=10). We did not detect differences in enzyme-inhibitor complexes between samples of controls, patients during remission and patients during an acute attack. Reconstitution with C1INH did not change this result. Next, we determined the potential to form enzyme-inhibitory complexes after complete in vitro activation of the plasma samples with a FXII trigger. In all samples, enzyme-C1INH levels increased after activation even in patients during an acute attack. However, the levels of FXIIa-C1INH, FXIa-C1INH and kallikrein-C1INH were at least 52% lower in samples taken during remission and 70% lower in samples taken during attack compared to samples from controls (p<0.05). Addition of C1INH after activation led to an increase in levels of FXIIa-C1INH and FXIa-C1INH (p<0.05), which were still lower than in controls (p<0.05), while the levels of kallikrein-C1INH did not change. These results are consistent with constitutive activation and attenuated depletion of the contact system and show that the ongoing activation of the contact system, which is present in HAE-C1INH patients both during remission and during acute attacks, is not associated with preferential generation of kallikrein over FXIa.

Activation of the contact system in patients with a first acute myocardial infarction.

INTRODUCTION: The contribution of the contact system to arterial thrombosis is unclear, results of clinical studies are conflicting. Particularly, little is known about the involvement of the contact system in the progression of arterial thrombosis. Therefore, we investigated the activation of the contact system during an acute myocardial infarction (AMI) and 3 and 6 months following the acute event. METHODS: Plasma of patients with a first AMI was collected on admission and 3 and 6 months after the AMI. The levels of complexes of activated factor XI (FXIa), FXIIa and kallikrein with C1 esterase inhibitor (C1INH) and the levels of complexes of FXIa with α1-antitrypsin (AT) were measured in these plasmas. Recurrent cardiovascular events were recorded during a one year period after the AMI. RESULTS: We observed that the levels of FXIa-C1INH were elevated during the acute phase compared to the steady-phase 3 and 6 months after the AMI. The levels of FXIa-AT, FXIIa-C1INH and kallikrein-C1INH did not change over time. The levels of FXIa-C1INH, FXIa-AT, FXIIa-C1INH and kallikrein-C1INH were not predictive for a recurrent event. CONCLUSION: We observed that during an AMI, the activation of FXI was increased. The levels of FXIIa-C1INH were not elevated, suggesting that activation of FXI during the acute phase did not result from contact activation. The levels of the enzyme inhibitor complexes were not predictive for a recurrent event one year after the first AMI.

Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation.

Platelets shed microparticles, which support haemostasis via adherence to the damaged vasculature and by promoting blood coagulation. We investigated mechanisms through which storage-induced microparticles might support blood coagulation. Flow cytometry was used to determine microparticle number, cellular origin and surface expression of tissue factor (TF), procoagulant phosphatidylserine (PtdSer) and glycoprotein (GP) Ib-alpha. The influence of microparticles on initiation and propagation of coagulation were examined in activated factor X (factor Xa; FXa) and thrombin generation assays and compared with that of synthetic phospholipids. About 75% of microparticles were platelet derived and their number significantly increased during storage of platelet concentrates. About 10% of the microparticles expressed functionally active TF, as measured in a FXa generation assay. However, TF-driven thrombin generation was only found in plasma in which tissue factor pathway inhibitor (TFPI) was neutralised, suggesting that microparticle-associated TF in platelet concentrates is of minor importance. Furthermore, 60% of all microparticles expressed PtdSer. In comparison with synthetic procoagulant phospholipids, the maximal rate of thrombin formation in TF-activated plasma was 15-fold higher when platelet-free plasma was titrated with microparticles. This difference could be attributed to the ability of microparticles to propagate thrombin generation by thrombin-activated FXI. Collectively, our findings indicate a role of microparticles in supporting haemostasis by enhancement of the propagation phase of blood coagulation.