The serpins (serine proteinase inhibitors) are a superfamily of proteins (350 -500 amino acids in size) that fold into a conserved structure and employ a unique suicide substrate-like inhibitory mechanism. The serpins were last reviewed in 1994 (1). More recent studies show: 1) an expanded distribution within the kingdoms of metazoa and plantae, as well as certain viruses, 2) a surprising effect on the covalently bound target proteinase, and 3) novel biochemical and biological functions.Most serpins inhibit serine proteinases of the chymotrypsin family. However, cross-class inhibitors have been identified. The viral serpin CrmA and, to a lesser extent, PI9 (SERPINB9) inhibit the cysteine proteinase, caspase 1 (2), and SCCA1

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Summary.  Hemostasis and fibrinolysis, the biological processes that maintain proper blood flow, are the consequence of a complex series of cascading enzymatic reactions. Serine proteases involved in these processes are regulated by feedback loops, local cofactor molecules, and serine protease inhibitors (serpins). The delicate balance between proteolytic and inhibitory reactions in hemostasis and fibrinolysis, described by the coagulation, protein C and fibrinolytic pathways, can be disrupted, resulting in the pathological conditions of thrombosis or abnormal bleeding. Medicine capitalizes on the importance of serpins, using therapeutics to manipulate the serpin–protease reactions for the treatment and prevention of thrombosis and hemorrhage. Therefore, investigation of serpins, their cofactors, and their structure–function relationships is imperative for the development of state‐of‐the‐art pharmaceuticals for the selective fine‐tuning of hemostasis and fibrinolysis. This review describes key serpins important in the regulation of these pathways: antithrombin, heparin cofactor II, protein Z‐dependent protease inhibitor, α1‐protease inhibitor, protein C inhibitor, α2‐antiplasmin and plasminogen activator inhibitor‐1. We focus on the biological function, the important structural elements, their known non‐hemostatic roles, the pathologies related to deficiencies or dysfunction, and the therapeutic roles of specific serpins.

The serine proteases sequentially activated to form a fibrin clot are inhibited primarily by members of the serpin family, which use a unique ␤-sheet expansion mechanism to trap and destroy their targets. Since the discovery that serpins were a family of serine protease inhibitors there has been controversy as to the role of conformational change in their mechanism. It now is clear that protease inhibition depends entirely on rapid serpin ␤-sheet expansion after proteolytic attack. The regulatory advantage afforded by the conformational mobility of serpins is demonstrated here by the structures of native and S195A thrombin-complexed heparin cofactor II (HCII). HCII inhibits thrombin, the final protease of the coagulation cascade, in a glycosaminoglycan-dependent manner that involves the release of a sequestered hirudin-like N-terminal tail for interaction with thrombin. The native structure of HCII resembles that of native antithrombin and suggests an alternative mechanism of allosteric activation, whereas the structure of the S195A thrombin-HCII complex defines the molecular basis of allostery. Together, these structures reveal a multistep allosteric mechanism that relies on sequential contraction and expansion of the central ␤-sheet of HCII.T he predominant protease inhibitors of the higher organisms, the serpins (1, 2), have evolved a complex mechanism that was revealed recently by the crystallographic structure of a serpin-protease complex (3). This dramatic mechanism amounts to a race between proteolysis and the incorporation of the serpin-reactive center loop into its main ␤-sheet (␤-sheet A). The dependence of the serpin inhibitory mechanism on ␤-sheet expansion renders serpins highly susceptible to both loss-offunction and gain-of-function diseases but additionally allows for a range of mechanisms for the modulation of serpin activity. Thus, serpins typically are found controlling tightly regulated physiological pathways such as blood coagulation. Antithrombin (AT) and heparin cofactor II (HCII) have independently (4, 5) evolved mechanisms by which they circulate in an inert state until activated by binding to cell surface glycosaminoglycans (GAGs). AT and HCII thus are allosterically activated toward factor Xa and thrombin, respectively, the final two proteases in the blood coagulation cascade.The structure and mechanism of activation of AT has been well characterized. Its fold is similar to that of the rest of the serpin family with the single and important exception that the reactive center loop is constrained through partial incorporation into ␤-sheet A (refs. 6 and 7; Fig. 1a). The native conformation of AT thus renders it incapable of forming a productive recognition complex (Michaelis complex) with factor Xa, and only as the result the conformational changes brought about through the interaction with a specific heparin pentasaccharide sequence does AT become an efficient inhibitor of factor Xa. Pentasaccharide binding results in secondary structural changes in the heparin binding region and the ex...

IntroductionThe link between cancer and venous thromboembolism (VTE) is referred to as Trousseau syndrome. Interestingly, different cancer types are associated with different rates of VTE, with pancreatic cancer having one of the highest rates. 1,2 A VTE risk-scoring model has been developed that stratifies ambulatory cancer patients undergoing chemotherapy into 3 VTE risk categories based on 5 parameters: (1) the site of the primary tumor, (2) prechemotherapy leukocyte count, (3) platelet count, (4) hemoglobin level, and (5) body mass index. 3 Recently, this model was expanded to include the biomarkers D-dimer and soluble P-selectin. 4 Another potential circulating biomarker of VTE risk in pancreatic cancer patients is microparticle (MP) tissue factor (TF). [5][6][7][8][9] Full-length TF (flTF) is a transmembrane protein that activates the coagulation cascade. 10 In addition, an alternatively spliced form of TF (asTF) has been identified that lacks a membrane anchor and therefore can be released as a soluble protein. 11 Increased TF expression is correlated with poor prognosis in pancreatic cancer. [12][13][14] Cultured human pancreatic tumor lines express variable levels of both flTF and asTF and release TF-positive MPs containing flTF into the culture medium. [14][15][16][17][18][19] In some patients with pancreatic cancer, high levels of TF-positive MPs are found in the circulation and, in a small pilot study, were predictive of VTE. [5][6][7]9,20 In a mouse model of human colorectal tumors, human TF protein is released into the circulation. 21 In nude mice bearing orthotopic human pancreatic tumors (L3.6pl) plasma levels of human TF protein were correlated with the levels of thrombin-antithrombin (TAT) complex, a marker of the activation of coagulation. 22 Further, plasma from these tumor-bearing mice was found to enhance thrombin generation in vitro in a human TF-dependent manner. 22 Another study found that human (SOJ-4) and mouse (PANC02) pancreatic cell lines expressed TF, and the investigators observed an accumulation of tumor-derived MPs at the site of thrombosis and increased thrombosis in a microvascular model. 18 The objective of the present study was to determine the role of tumor-derived TF in the activation of coagulation and thrombosis in a xenograft mouse model of human pancreatic tumors. We found that only TF-positive tumors activated coagulation and that this activation was abolished by inhibition of human TF. Two TF-positive pancreatic tumor cell lines activated coagulation, but only one had detectable levels of circulating TF-positive MPs, which suggested that activation of coagulation was due to TF expression by the tumor itself rather than to TF on the MPs. Mice with elevated levels of TF-positive MPs exhibited increased thrombosis in a saphenous vein model, but not in an inferior vena cava (IVC) stenosis model. Methods Cell linesHuman pancreatic (MIAPaCa-2 [CRL-1420] Abs and proteinsPE-labeled mouse IgG control (#555574), mouse anti-human TF (#550312) and FITC-conjugated anti-human MUC...

Epidemiologic studies have correlated elevated plasma fibrinogen (hyperfibrinogenemia) with risk of cardiovascular disease and arterial and venous thrombosis. However, it is unknown whether hyperfibrinogenemia is merely a biomarker of the proinflammatory disease state or is a causative mechanism in the etiology. We raised plasma fibrinogen levels in mice via intravenous infusion and induced thrombosis by ferric chloride application to the carotid artery (high shear) or saphenous vein (lower shear); hyperfibrinogenemia significantly shortened the time to occlusion in both models. Using immunohistochemistry, turbidity, confocal microscopy, and elastometry of clots produced in cell and tissue factor-initiated models of thrombosis, we show that hyperfibrinogenemia increased thrombus fibrin content, promoted faster fibrin formation, and increased fibrin network density, strength, and stability. Hyperfibrinogenemia also increased thrombus resistance to tenecteplase-induced thrombolysis in vivo. These data indicate that hyperfibrinogenemia directly promotes thrombosis and thrombolysis resistance and does so via enhanced fibrin formation and stability. These findings strongly suggest a causative role for hyperfibrinogenemia in acute thrombosis and have significant implications for thrombolytic therapy. Plasma fibrinogen levels may be used to identify patients at risk for thrombosis and inform thrombolytic administration for treating acute thrombosis/thromboembolism. IntroductionElevated plasma fibrinogen is associated with risk of cardiovascular disease and arterial and venous thrombosis. [1][2][3][4][5][6][7][8][9] Several studies have detected dose effects, with increased risk of death or thrombosis in subjects with the highest plasma fibrinogen concentrations. [6][7][8][9] The Framingham 7 and Fragmin During Instability in Coronary Artery Disease 8 studies positively correlated fibrinogen levels with risk of cardiovascular disease and incidence of death and/or myocardial infarction, respectively. The Leiden Thrombophilia Study showed that persons with elevated fibrinogen levels (4.0-4.9 vs Ͻ 3.0 mg/mL, 130%-160% of normal) have an adjusted odds ratio for venous thrombosis of 1.6, whereas persons with Ն 5 mg/mL fibrinogen (Ն 170% of normal) have a 4-fold higher thrombotic risk, even after adjusting for C-reactive protein levels. 9 These epidemiologic studies suggest that elevated fibrinogen is an independent risk factor for both arterial and venous thrombosis and therefore a potential diagnostic and therapeutic target for predicting and reducing thrombosis.Importantly, however, epidemiologic studies have not and cannot show a causal relationship between fibrinogen and disease etiology. 2,10,11 Fibrinogen levels increase with age, inflammatory processes, hematocrit, hypertension, glucose intolerance, cigarette smoking, and adiposity, and high fibrinogen levels increase plasma viscosity, a demonstrated risk factor for coronary heart disease. 5,6,12 These potential confounders have not permitted distinction between fibr...

Protein C inhibitor (PCI),

While we are still learning more about COVID-19, caused by the novel SARS-CoV-2 virus, finding alternative and already available methods to reduce the risk and severity of the disease is paramount. One such option is vitamin D, in the form of vitamin D 3 (cholecalciferol) supplementation, due to its potential antiviral properties. It has become apparent that older individuals have a greater risk of developing severe COVID-19, and compared to younger adults, the elderly have lower levels of vitamin D due to a variety of biological and behavioral factors. Older adults are also more likely to be diagnosed with Parkinson's disease (PD), with advanced age being the single greatest risk factor. In addition to its immune-system-modulating effects, it has been suggested that vitamin D supplementation plays a role in slowing PD progression and improving PD-related quality of life. We completed a review of the literature to determine the relationship between vitamin D, PD, and COVID-19. We concluded that the daily supplementation of 2000-5000 IU/day of vitamin D 3 in older adults with PD has the potential to slow the progression of PD while also potentially offering additional protection against COVID-19.