The kinetic parameters of the conversion of prothrombin into thrombin by activated clotting factor X (factor Xa) have been determined in the absence and presence of Ca2+, phospholipid (phosphatidyl serine/phosphatidylcholine vesicles) and activated blood clotting factor V (factor Va). In free solution the Km for prothrombin is 298 μM which is well above its plasma concentration of 4μM. Under these conditions the Vmax of thrombin formation is 1.25 Moles min-1 Mole Xa -1. When phospholipid is present the km for prothrombin drops to 0.1μM while the Vmax is only slightly affected (3 Moles min-1 Mo Le Xa -1). For the complete prothrombin activating complex consisting of factor Xa, factor Va, Ca2+ and phospholipids the kinetic constants greatly favour thrombin formation. A for prothrombin of 0.26μM and a Vmax of 2130 Moles min-1 Mole xa -1 are measured under these conditions. These results help to elucidate the role of phospholipid and factor Va in prothrombin activation. The earlier observed rate enhancements caused by phospholipid and factor Va are explained as effects on the Km for prothrombin and the Vmax of thrombin formation, respectively. The changes of the kinetic parameters for prothrombinase complexes of various composition will be considered with respect to the function of the accessory components in the mechanism of prothrombin activation. Implications of these data for in vivo blood coagulation will be discussed.

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 kallikreinkinin 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.

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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 dosedependent 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 C␥2) 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/factorVIIa 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 PLC␥2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi. (Blood. 2009; 114:881-890) IntroductionVascular injury leads to exposure of hemostatically active components, such as tissue factor and collagen, to the bloodstream. For a long time, it has been considered that exposed tissue factor is the main vascular activator of the coagulation cascade. In the extrinsic pathway of coagulation, de-encrypted tissue factor complexes with circulating factor (F)VII(a), which activates a multistep cascade of serine proteases to form thrombin and fibrin. 1 The exposed collagen is thought to function as a substrate for the adhesion and activation of platelets via the glycoprotein VI (GPVI) receptor, which evokes an important signal transduction pathway in platelets. 2 Independently of tissue factor, the second intrinsic coagulation pathway is initiated by activation of plasma FXII (Hageman factor). Proteolytically active FXIIa mediates the sequential activation of the serine proteases, FXI and FIX, which also lead to thrombin generation. Until recently, the physiologic relevance of this pathway has remained obscure, as the initial trigger in vivo was not known. On the other hand, in vitro the intrinsic system is easily triggered in plasma using negatively charged materials, such as kaolin, glass, and ellagic acid. Common clotting tests, such as the activated partial thromboplastin time (aPTT), rely on activation of the intrinsic coagulation pathway. In the 1980s, it was proposed that polyanionic vascular components, such as cerebroside sulfates and glycosaminoglycans, function to stimulate FXII activation. 3,4 A similar role for collagen was suggested, 5,6 but this was refuted by other authors. [7][8][9] Recent studies with mice have greatly increased the inte...

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 (nϭ27) 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] Key Words: atherosclerosis Ⅲ hypercoagulability Ⅲ immunohistochemistry Ⅲ plaque Ⅲ thrombosis A therosclerosis is widely recognized as a chronic inflammatory disease. 1 Rupture of an atherosclerotic plaque is considered the predominant underlying cause of acute atherothrombotic events such as myocardial infarction, ischemic stroke, and vascular death. A close relation between blood coagulation and atherosclerosis 2,3 is supported by studies revealing the presence of specific coagulation proteins within an atherosclerotic lesion. Tissue factor (TF) and factor (F) VII, of which the complex is the principal initiator of coagulation in vivo, are expressed on macrophages and vascular smooth muscle cells (SMC) within the arterial vessel wall and atherosclerotic lesion. 4,5 Both proteins potentially participate in multiple proatherogenic processes such as migration and proliferation of SMC, 6 inflammation, and angiogenesis. 7 In addition to the single effects of each protein, the local interaction between macrophage/SMC-derived TF and FVII may provide a catalytic complex for subsequent generation of thrombin and fibrin, of which the latter is also detectable in atherosclerotic lesions. 8,9 The procoagulant condition of the atherosclerotic lesion may be further enhanced by the presence of various proinflammatory cytokines (eg, tumor necrosis factor-␣, interleukin-1 10 ), which may downregulate local expression of anticoagulant proteins such as thrombomodulin and the endothelial protein C receptor on endothelial cells. 11 Received September 4, 2009; accepted June 28, 2010 Clinical Perspective on p 830Thrombin, a key enzyme in blood coagulation, may also play a critical role in many processes related to the development, progression, and atherothrombotic potential of atherosclerotic plaques. 12 Direct evidence for the role of thrombin in the atherogenic process comes from experiments showing reduced progression of atherosclerosis in apolipoprotein E Ϫ/Ϫ mice on pharmacological inhibition of thrombin. 13 Moreover, decreased expression of TF pathway inhibitor (TFPI) on an apolipoprotein E Ϫ/Ϫ background increased the atheroscle...