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 prothrombindeficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and ␣-FXIIa, but not ␤-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 IntroductionBlood coagulation culminates in the formation of fibrin, which binds platelets and forms a clot. Fibrin is formed from fibrinogen via cleavage of 2 fibrinopeptides from the A␣-and B␤-chains N-termini, located in the E-region, by thrombin. 1 Fibrinopeptide cleavage exposes binding sites for complementary sites in the D-region, triggering polymerization and the production of protofibrils. Protofibrils aggregate laterally to form fibers, which branch out and form a 3-dimensional network. 2 There is increasing evidence that the structure of fibrin regulates thrombosis. Dense fibrin clots with small pores and increased fiber density are more resistant to lysis. 3 Structural characteristics affect the mechanical properties of fibrin. 4 Both venous and arterial thrombosis has been associated with the formation of an altered fibrin network. [5][6][7][8][9][10] The role of factor (F)XII in hemostasis has long been contested because deficiency in FXII, unlike deficiencies of other coagulation factors, does not lead to bleeding diathesis in humans 11 or in mice. 12 However, recent in vivo data show that FXII deficiency or inhibition in rodent models reduces thrombus formation while maintaining normal hemostasis. [12][13][14][15] These findings indicate the existence of FXII-related mechanisms that are preferentially involved in thrombosis but not hemostasis.Contact activation is triggered by the binding of FXII (80 kDa) to a negatively charged surface and involves the formation of ␣-FXIIa via autocatalysis. Bound ␣-FXIIa converts prekallikrein into kallikrein. Kallikrein can further convert ␣-FXIIa to ␤-FXIIa by an additional cleavage at R334-N335. ␣-FXIIa consists of a heavy and light chain that are disulphide linked (80 kDa), whereas ␤-FXIIa (28 kDa) lacks the heavy chain and loses its capacity to bind to negatively charged surfaces. 16 The N-terminal region of FXII (␣-FXIIa heavy chain) shows strong homology with tissuetype plasminogen activator (tPA), with the presence of fibr...

Hemostasis requires conversion of fibrinogen to fibrin fibers that generate a characteristic network, interact with blood cells, and initiate tissue repair. The fibrin network is porous and highly permeable, but the spatial arrangement of the external clot face is unknown. Here we show that fibrin transitioned to the blood-air interface through Langmuir film formation, producing a protective film confining clots in human and mouse models. We demonstrated that only fibrin is required for formation of the film, and that it occurred in vitro and in vivo. The fibrin film connected to the underlying clot network through tethering fibers. It was digested by plasmin, and formation of the film was prevented with surfactants. Functionally, the film retained blood cells and protected against penetration by bacterial pathogens in a murine model of dermal infection. Our data show a remarkable aspect of blood clotting in which fibrin forms a protective film covering the external surface of the clot, defending the organism against microbial invasion.

IntroductionPlatelet function testing with flow cytometry has additional value to existing platelet function testing for diagnosing bleeding disorders, monitoring anti-platelet therapy, transfusion medicine and prediction of thrombosis. The major challenge is to use this technique as a diagnostic test. The aim of this study is to standardize preparation, optimization and validation of the test kit and to determine reference values in a population of 129 healthy individuals.MethodsPlatelet function tests with 3 agonists and antibodies against P-selectin, activated αIIbβ3 and glycoprotein Ib (GPIb), were prepared and stored at -20°C until used. Diluted whole blood was added and platelet activation was quantified by the density of activation markers, using flow cytometry. Anti-mouse Ig κ particles were included to validate stability of the test and to standardize results. Reference intervals were determined.ResultsBlood stored at room temperature (RT) for up to 4h after blood donation and preheated/tested at 37°C resulted in stable results (%CV<10%), in contrast to measuring at RT. The intra-assay %CV was <5%. Incubation of anti-mouse Ig κ particles with antibodies stored for up to 12 months proved to give a stable fluorescence. The inter-individual variation measured in the 129 individuals varied between 23% and 37% for P-selectin expression and αIIbβ3 activation, respectively.ConclusionsThe current study contributes to the translation of flow cytometry based platelet function testing from a scientific tool to a diagnostic test. Platelet function measurements, using prepared and stored platelet activation kits, are reproducible if executed at 37°C. The reference ranges can be validated in clinical laboratories and ongoing studies are investigating if reduced platelet reactivity in patients with bleeding complications can be detected.

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.

Background Assays measuring thrombin generation (TG) in plasma increasingly gained attention in the field of thrombosis and hemostasis. Adaptation of the method enabled the measurement of TG in whole blood (WB). Despite their potential, TG assays did not reach the stage of universal clinical application, partly because of the absence of normal ranges. Our study aimed to accurately determine normal ranges and interindividual variability of TG and correlate results with coagulation factor levels, sex, and oral contraceptive usage. Methods The study protocol was evaluated by the local medical ethical board. In total, 129 healthy volunteers gave full informed consent. Normal ranges of TG in platelet-poor plasma (PPP), platelet-rich plasma (PRP), and WB were determined according to CLSI guidelines. Results Our study is the first to measure normal ranges of TG in PPP, PRP, and WB in a large healthy cohort. Significant correlations were found between TG in plasma and WB. Interindividual variability of TG in WB was comparable to that of plasma. Oral contraceptive use increased TG in PPP, PRP, and WB. The inhibitory effect of thrombomodulin on TG was significantly lower in females than in males. This effect was more pronounced upon oral contraceptive use. Primary clotting factor determinants for TG parameters depended on the tissue factor concentration, but were similar in WB and plasma. Conclusions Establishing normal ranges for TG brings us 1 step closer to clinical use. Good correlations between plasma and WB (including clotting factor determinants for TG) suggest that WB TG can be reliably used in clinic.

Background Fluorogenic thrombin generation (TG) assays are commonly used to determine global coagulation phenotype in plasma. Whole blood (WB)‐TG assays reach one step closer to physiology by involving the intrinsic blood cells, but erythrocytes cause variable quenching of the fluorescence signals, hampering its routine application. Objective To develop a new assay for continuous WB‐TG measurement. Methods In the new WB‐TG assay, the erythrocyte‐caused distortion of signal was solved by continuously mixing the sample during the measurement. The assay was validated by evaluating the reproducibility and comparing with the paper‐based WB‐TG assay. Reconstituted human blood and WB from 119 healthy donors was tested to explore the influences of hematocrit and platelet count on TG. Results This novel WB‐TG assay showed good reproducibility while being less affected by contact activation compared with the previous paper‐based assay. Reconstitution experiments showed that the lag time of TG was shortened by the addition of platelets but not erythrocytes. Increasing hematocrit strongly augmented the peak thrombin, even in the presence of high platelet counts. The lag time and peak of WB‐TG of 119 healthy donors were positively related to erythrocyte count after adjusting for age, sex, and oral contraceptive use with multiple linear regression analyses. The reference range and interindividual variation of WB‐TG were determined in the healthy cohort. Conclusions A novel WB‐TG assay was developed, which is a straightforward tool to measure the involvement of platelets and erythrocytes in TG and may assist the research of blood cell‐associated coagulation disorders.

Background and Aims Patients with cirrhosis have a rebalanced hemostasis, often with normal or elevated thrombin‐generating (TG) capacity in plasma. Whole blood (WB) TG allows faster determination and, importantly, includes the influence of all circulating blood cells. We aimed to study the TG profile of patients with cirrhosis in WB and in platelet poor plasma. Methods Thrombin‐generating capacity in WB and plasma were assessed with a near‐patient WB‐TG assay and the calibrated automated thrombinography assay, respectively. TG assays were tested in presence and absence of thrombomodulin. Conventional coagulation tests were also performed. Results Thirty‐four patients with cirrhosis and twenty‐two controls were analyzed. Compared with controls, patients had substantially deranged results in conventional coagulation tests. Comparable WB‐TG capacity (endogenous thrombin potential until peak, ETPp) but significantly lower peak thrombin were found in patients, and these results persisted when thrombomodulin was present. TG of the patients was more resistant to thrombomodulin than controls in both WB and plasma, although the inhibitory effect of thrombomodulin was drastically weaker in WB than in plasma. The peak of WB‐TG in patients correlated moderately with their hematocrit and platelet count. Significant correlations were found between TG results in WB and plasma. Conclusions The WB‐TG assay shows a normal to hypocoagulable state in patients with cirrhosis with a decreased anticoagulant activity of TM compared to plasma‐TG. The clinical value of this assay needs further validation.

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.