Effective hemostasis relies on the timely formation of-thrombin via prothrombi-nase, a Ca 2-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrom-bin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when pro-thrombin is processed on the activated platelet surface, the cleavage of prothrom-bin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin-or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating pre-thrombin-2, with-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma-or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC 50 0.3M), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagu-lant activity expressed by activated plate-lets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin. (Blood. 2011;117(5): 1710-1718) Introduction The activation of prothrombin to-thrombin is a critical step in the response to vascular injury. The generation of-thrombin is achieved through the action of prothrombinase, which is composed of the serine protease factor Xa and its nonenzymatic cofactor, factor Va, assembled on an appropriate membrane surface in the presence of Ca 2 ions. 1 In the physiologic setting, this surface is provided by the activated platelet. 2 Relative to the activity of factor Xa alone, incorporation of factor Xa into prothrombinase accelerates the rate of prothrombin cleavage by 5 orders of magnitude, 3 and both factor Va and the membrane surface are critical in this rate amplification, as removal of either component results in a substantial decrease in the rate of prothrombin cleavage. 3 Indeed, deficiencies or disorders of any component of this complex result in severe bleeding diatheses. 4-6 Prothrombin, the substrate for prothrombinase, consists of 4 domains: fragment 1, fragment 2, and the A and B chains of-thrombin (Figure 1). 1 Prothrombin is proteolytically activated to-thrombin by cleavage on the C-terminal side of 2 specific residues: Arg271 (located between fragment 2 and the A chain) and Arg320 (located between the A and B chains). Initial cleavage at Arg271 results in the generation of prethrombin-2, an inactive intermediate, and the release of fragment 1.2 (F1.2). Subsequent cleavage at Arg320 converts prethrombin-2 to-thrombin. 7 Alternatively , cleavage may occur first at Arg320, leading to the generation of the enzymatically active interme...

Background:The key source of prothrombin activation in vivo is prothrombinase assembled on the activated platelet surface. Results: Platelet-associated prothrombinase utilizes the prethrombin-2 pathway of prothrombin activation and a concerted enzyme mechanism. Conclusion: Platelet-associated prothrombinase activates prothrombin with a concerted mechanism in which no anticoagulant intermediates are released. Significance: Platelet-associated prothrombinase promotes coagulation by avoiding the release of catalytically active meizothrombin.

The generation of proteolyzed prothrombin species by preassembled prothrombinase in phospholipid-coated glass capillaries was studied at physiologic shear rates (100-1000 s(-1)). The concentration of active thrombin species (α-thrombin and meizothrombin) reaches a steady state, which varies inversely with shear rate. When corrected for shear rate, steady-state levels of active thrombin species exhibit no variation and a Michaelis-Menten analysis reveals that chemistry of this reaction is invariant between open and closed systems; collectively, these data imply that variations with shear rate arise from dilutional effects. Significantly, the major products observed include nonreactive species arising from the loss of prothrombin's phospholipid binding domain (des F1 species). A numerical model developed to investigate the spatial and temporal distribution of active thrombin species within the capillary reasonably approximates the observed output of total thrombin species at different shears; it also predicts concentrations of active thrombin species in the wall region sufficient to account for observed levels of des FI species. The predominant feedback formation of nonreactive species and high levels of the primarily anticoagulant intermediate meizothrombin (∼40% of total active thrombin species) may provide a mechanism to prevent thrombus propagation downstream of a site of thrombosis or hemorrhage.

We use laser photoacoustic spectroscopy to obtain vibrational overtone spectra in the regions of four and five quanta of O-H stretch (4ν OH and 5ν OH ) for gas-phase methyl (MeOOH), ethyl (EtOOH), and tert-butyl hydroperoxide (t-BuOOH). Integrated cross sections for the main peak due to O-H stretch excitation to 4ν OH are similar for all three hydroperoxides (∼2 × 10 -21 cm 2 molecule -1 cm -1 ); cross sections for excitation to and 5ν OH are an order of magnitude smaller. Interpretation of spectral features using a previously reported vibration-torsion model for ROOH molecules, where excitation of torsional motion about the O-O bond accompanies vibrational excitation, yields plausible fits to the spectra. Simulations with the vibration-torsion model and ab initio calculations suggest barriers for torsional motion about the O-O bond to be higher in t-BuOOH than in MeOOH and EtOOH, with a trend of increasing trans torsional barrier height with increasing size of the R group.

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). Missense variants render PAI-1 non-functional through misfolding, leading to its turnover as a protease substrate, or to a more rapid transition to the latent/inactive state. Deep mutational scanning was performed to evaluate the impact of amino acid sequence variation on PAI-1 inhibition of uPA using an M13 filamentous phage display system. Error prone PCR was used to construct a mutagenized PAI-1 library encompassing ~ 70% of potential single amino acid substitutions. The relative effects of 27% of all possible missense variants on PAI-1 inhibition of uPA were determined using high-throughput DNA sequencing. 826 missense variants demonstrated conserved inhibitory activity while 1137 resulted in loss of PAI-1 inhibitory function. The least evolutionarily conserved regions of PAI-1 were also identified as being the most tolerant of missense mutations. The results of this screen confirm previous low-throughput mutational studies, including those of the reactive center loop. These data provide a powerful resource for explaining structure–function relationships for PAI-1 and for the interpretation of human genomic sequence variants.