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...

This study was designed to differentiate the contributions of hyperandrogenism, insulin resistance (IR), and body weight to the development of endothelial dysfunction in polycystic ovary syndrome and determine the effectiveness of insulin sensitization and antiandrogenic therapy after the establishment of vascular and metabolic dysfunction using a rat model of polycystic ovary syndrome. We hypothesized that the observed endothelial dysfunction was a direct steroidal effect, as opposed to changes in insulin sensitivity or body weight. Prepubertal female rats were randomized to the implantation of a pellet containing DHT or sham procedure. In phase 1, DHT-exposed animals were randomized to pair feeding to prevent weight gain or metformin, an insulin-sensitizing agent, from 5 to 14 weeks. In phase 2, DHT-exposed animals were randomized to treatment with metformin or flutamide, a nonsteroidal androgen receptor blocker from 12 to 16 weeks. Endothelial function was assessed by the vasodilatory response of preconstricted arteries to acetylcholine. Serum steroid levels were analyzed in phase 1 animals. Fasting blood glucose and plasma insulin were analyzed and homeostasis model assessment index calculated in all animals. Our data confirm the presence of endothelial dysfunction as well as increased body weight, hypertension, hyperinsulinemia, and greater IR among DHT-treated animals. Even when normal weight was maintained through pair feeding, endothelial dysfunction, hyperinsulinemia, and IR still developed. Furthermore, despite weight gain, treatment with metformin and flutamide improved insulin sensitivity and blood pressure and restored normal endothelial function. Therefore, the observed endothelial dysfunction is most likely a direct result of hyperandrogenism-induced reductions in insulin sensitivity, as opposed to weight gain.

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Perivascular adipose tissue (PVAT) contributes to vasoregulation. The role of this adipose tissue bed in pregnancy has not been examined. Here, we tested the hypothesis that PVAT in pregnant rats decreases resistance artery tone. Mesenteric arteries from nonpregnant (NP) and late pregnant (LP) rats were exposed to phenylephrine (PHE) or KCl in the presence (þ) versus absence (À) of PVAT. The LP PVAT(þ) vessels showed a 44% decrease in sensitivity to PHE in the presence of PVAT. There was no attenuation of the contractile response to KCl when PVAT was present. The LP arteries perfused with LP or NP PVAT underwent vasodilation; unexpectedly, NP vessels in the presence of PVAT from LP rats sustained a 48% vasoconstriction. The PVAT attenuates vasoconstriction by a mechanism that involves hyperpolarization. The vasoconstriction observed when nonpregnant vessels were exposed to pregnant PVAT suggests pregnant vessels adapt to the vasoconstricting influence of pregnant PVAT.

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