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.