Poly-P as Modulator of Hemostasis, Thrombosis, and Inflammation

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-1-SCI-1
Author(s):  
James H. Morrissey
Keyword(s):  
Mammalian Cells ◽  
Research Funding ◽  
Blood Clotting ◽  
Anticoagulant Activity ◽  
Fibrin Clot ◽  
Factor V ◽  
Linear Polymers ◽  
Clotting System ◽  
Subsequent Work ◽  
Dense Granules

Polyphosphate (polyP), consisting of linear polymers of inorganic phosphates, is ubiquitous in biology. PolyP metabolism has been most extensively explored in microbes, but until very recently, roles for polyP in mammalian cells have been poorly understood. In 2004, polyP was shown to be a major component of dense granules in human platelets, and to be secreted upon platelet activation.1 In 2006, we demonstrated that polyP is a potent modulator of the blood clotting system.2 Subsequent work from our lab and others has shown that polyP accelerates blood clotting and slows fibrinolysis, in a manner that is highly dependent on polymer length.3 Long-chain polyP (present in infectious microorganisms) potently triggers the contact pathway and may participate in innate immunity/host responses to pathogens. PolyP of the size secreted by platelets (60 to 100 phosphates long) accelerates factor V activation, abrogates the anticoagulant activity of tissue factor pathway inhibitor, enhances fibrin clot structure, and greatly accelerates factor XI activation by thrombin. PolyP also enhances the proinflammatory activity of histones and inhibits complement. PolyP is a potential antithrombotic drug target, with a novel mechanism of action and possibly fewer bleeding side-effects compared to conventional anticoagulant drugs.4 1. Ruiz FA, Lea CR, Oldfield E, Docampo R. Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. J Biol Chem . 2004;279(43):44250-44257. 2. Smith SA, Mutch NJ, Baskar D, Rohloff P, Docampo R, Morrissey JH. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci U S A . 2006;103(4):903-908. 3. Morrissey JH, Smith SA. Polyphosphate as modulator of hemostasis, thrombosis, and inflammation. J Thromb Haemost . 2015;13 Suppl 1:S92-97. 4. Travers RJ, Shenoi RA, Kalathottukaren MT, Kizhakkedathu JN, Morrissey JH. Nontoxic polyphosphate inhibitors reduce thrombosis while sparing hemostasis. Blood . 2014;124(22):3183-3190. Disclosures Morrissey: Novo Nordisk: Consultancy, Honoraria, Research Funding; rEVO Biologics: Consultancy, Honoraria; Paul, Weiss, Rifkind, Wharton & Garrison LLP: Consultancy; Cayuga Pharmaceuticals: Consultancy; PrevThro Pharmaceuticals: Equity Ownership; Kerafast, Inc.: Research Funding.

scholarly journals The Blood Clotting Properties of Rabbit Peritoneal Leukocytes in Vitro

1967 ◽  
Vol 17 (01/02) ◽  
pp. 222-236 ◽  
Author(s):  
S. I Rapaport ◽  
P. F Hjort
Keyword(s):  
Blood Clotting ◽  
Anticoagulant Activity ◽  
In Vitro Study ◽  
Factor V ◽  
Clotting Factors ◽  
Clotting System ◽  
Procoagulant Effect ◽  
Peritoneal Leukocytes ◽  
Thromboplastic Activity

SummaryA systematic in vitro study has been carried out of the blood clotting properties of rabbit peritoneal leukocytes. Rabbit heterophils have been shown to possess weak but definite tissue thromboplastic activity. They also contain an anticoagulant activity which is active in the intrinsic clotting system, in the extrinsic clotting system with brain thromboplastin, and in clotting systems with Russell’s viper venom. When the thromboplastic activity of the white cell itself initiates clotting, the anticoagulant is much less effective and the procoagulant effect of the WBC suspension predominates. Rabbit heterophils do not bind plasma clotting factors, do not activative factor V, and, under our conditions, do not precipitate fibrinogen from normal plasma, from plasma exposed to traces of thrombin in vitro, or from plasma of rabbits given endotoxin.The relation of these findings to the role of the granulocyte in the pathogenesis of the generalized Shwartzman reaction has been discussed.

scholarly journals Polyphosphate exerts differential effects on blood clotting, depending on polymer size

Blood ◽  
2010 ◽  
Vol 116 (20) ◽  
pp. 4353-4359 ◽  
Author(s):  
Stephanie A. Smith ◽  
Sharon H. Choi ◽  
Rebecca Davis-Harrison ◽  
Jillian Huyck ◽  
John Boettcher ◽  
...  
Keyword(s):  
Blood Clotting ◽  
Fibrin Clot ◽  
Host Responses ◽  
Coagulation Cascade ◽  
Factor V ◽  
Differential Effects ◽  
Activated Platelets ◽  
Contact Pathway ◽  
Anticoagulant Function ◽  
Clot Structure

AbstractPolyphosphate, a linear polymer of inorganic phosphate, is secreted by activated platelets and accumulates in many infectious microorganisms. We recently showed that polyphosphate modulates the blood coagulation cascade at 3 steps: it triggers the contact pathway, it accelerates factor V activation, and it enhances fibrin polymerization. We now report that polyphosphate exerts differential effects on blood clotting, depending on polymer length. Very long polymers (≥ 500mers, such as those present in microorganisms) were required for optimal activation of the contact pathway, while shorter polymers (∼ 100mers, similar to the polymer lengths released by platelets) were sufficient to accelerate factor V activation and abrogate the anticoagulant function of the tissue factor pathway inhibitor. Optimal enhancement of fibrin clot turbidity by polyphosphate required ≥ 250mers. Pyrophosphate, which is also secreted by activated platelets, potently blocked polyphosphate-mediated enhancement of fibrin clot structure, suggesting that pyrophosphate is a novel regulator of fibrin function. In conclusion, polyphosphate of the size secreted by platelets is very efficient at accelerating blood clotting reactions but is less efficient at initiating them or at modulating clot structure. Microbial polyphosphate, which is highly procoagulant, may function in host responses to pathogens.

scholarly journals Polyphosphate: an ancient molecule that links platelets, coagulation, and inflammation

Blood ◽  
2012 ◽  
Vol 119 (25) ◽  
pp. 5972-5979 ◽  
Author(s):  
James H. Morrissey ◽  
Sharon H. Choi ◽  
Stephanie A. Smith
Keyword(s):  
Blood Clotting ◽  
Molecular Mechanisms ◽  
Fibrin Clot ◽  
Human Platelets ◽  
Factor V ◽  
Inorganic Polyphosphate ◽  
Contact Pathway ◽  
Anticoagulant Function ◽  
Clot Structure

AbstractInorganic polyphosphate is widespread in biology and exhibits striking prohemostatic, prothrombotic, and proinflammatory effects in vivo. Long-chain polyphosphate (of the size present in infectious microorganisms) is a potent, natural pathophysiologic activator of the contact pathway of blood clotting. Medium-chain polyphosphate (of the size secreted from activated human platelets) accelerates factor V activation, completely abrogates the anticoagulant function of tissue factor pathway inhibitor, enhances fibrin clot structure, and greatly accelerates factor XI activation by thrombin. Polyphosphate may have utility as a hemostatic agent, whereas antagonists of polyphosphate may function as novel antithrombotic/anti-inflammatory agents. The detailed molecular mechanisms by which polyphosphate modulates blood clotting reactions remain to be elucidated.

Short Polyphosphates Inhibit the Procoagulant Activities of Long-Chain Polyphosphate: A Novel Role for Platelet-Derived Pyrophosphate?.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1140-1140
Author(s):  
Stephanie A. Smith ◽  
James H. Morrissey
Keyword(s):  
Factor Xa ◽  
Fibrin Clot ◽  
Short Chain ◽  
Factor V ◽  
Fibrin Polymerization ◽  
Dense Granules ◽  
Contact Pathway ◽  
Clot Structure ◽  
Long Chain ◽  
Potent Inhibitory Activity

Abstract Abstract 1140 Introduction: Inorganic polyphosphates are negatively charged, linear phosphate polymers that influence hemostasis via accelerating factor V activation, triggering the contact pathway, and enhancing fibrin polymerization. The latter two effects require long-chain polyphosphates for optimal activity (>1000mers for activating the contact pathway and >250mers for enhancing fibrin polymerization). We explored whether short polyphosphates could inhibit the procoagulant effects of long polyphosphate polymers. Methods: Polyphosphate was size-fractionated by gel electrophoresis. “Long-chain polyphosphate” (LCP) was a heterogeneous polyphosphate preparation containing polymers ranging from 400 to several thousand phosphates long. Short-chain polyphosphate preparations were narrow fractions of homogeneous size. We also tested additional phosphate-containing molecules including ADP, ATP, monophosphate, pyrophosphate (PPi), and triphosphate. Clotting assays were performed using pooled normal plasma spiked with 0–20 μM LCP and 0–500 μM small (poly)phosphate; clotting was initiated by factor Xa plus CaCl2, or by CaCl2 alone (contact pathway). Fibrin was formed by clotting 2.6 mg/ml fibrinogen with 10 nM thrombin in the presence of 0–500 μM small (poly)phosphate plus 150 μM LCP and CaCl2. Results: Small polyphosphates (size range 23–83mer) reduced the ability of LCP to trigger the contact pathway of blood clotting, while monophosphate, PPi, triphosphate, ADP, and ATP all failed to influence the procoagulant activity of LCP. None of the small (poly)phosphates antagonized the ability of LCP to enhance factor V activation. Interestingly, monophosphate, PPi and triphosphate all inhibited the ability of LCP to enhance fibrin clot structure, with PPi being the most potent. Although ADP and ATP contain di- and tri-phosphates, they did not recapitulate the potent inhibitory activity of PPi. On the other hand, PPi had no measurable effect on the turbidity of fibrin clots formed in the absence of LCP. Conclusions: Short-chain polyphosphates inhibit the ability of LCP to initiate the contact pathway of coagulation. Platelet dense granules contain abundant PPi which is secreted in response to platelet agonists, although biological roles for platelet-secreted PPi are unclear. We propose that PPi is a novel modulator of fibrin clot structure, acting to regulate the effects of longer-chain polyphosphates on fibrin fibril formation. Disclosures: Smith: University of Illinois: Patents & Royalties. Morrissey:University of Illinois: Patents & Royalties.

scholarly journals Secretion of a proteolytic anticoagulant by Ancylostoma hookworms.

1983 ◽  
Vol 157 (5) ◽  
pp. 1594-1603 ◽  
Author(s):  
P J Hotez ◽  
A Cerami
Keyword(s):  
Human Plasma ◽  
Synthetic Peptide ◽  
Proteolytic Enzyme ◽  
Blood Clotting ◽  
Anticoagulant Activity ◽  
Fibrin Clot ◽  
Peptide Substrate

Hookworms of the genus Ancylostoma secrete an anticoagulant that both inhibits the clotting of human plasma and promotes fibrin clot dissolution. This anticoagulant activity is attributable to a 36,000 dalton proteolytic enzyme. The protease can degrade fibrinogen into five smaller polypeptides that intrinsically have anticoagulating properties, covert plasminogen to a mini-plasminogen-like molecule, and hydrolyze a synthetic peptide substrate with specificity for elastolytic enzymes. It is hypothesized that the parasite uses this enzyme to prevent blood clotting while feeding on villous capillaries.

Size Matters: Differential Effects of RNA and Polyphosphate on Blood Clotting

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3074-3074
Author(s):  
Stephanie A. Smith ◽  
James H. Morrissey
Keyword(s):  
Metal Ion ◽  
Blood Clotting ◽  
Factor Xa ◽  
Fibrin Clot ◽  
Factor V ◽  
Clotting Time ◽  
Yeast Trna ◽  
Clotting Cascade ◽  
Contact Pathway ◽  
Clot Structure

Abstract Introduction: Recently, both inorganic polyphosphate (Smith et al., PNAS103:903-8, 2006) and RNA (Kannemeier et al., PNAS104:6388–93, 2007) have been proposed as (patho)physiologic activators of the contact pathway in blood clotting. We also recently showed that polyphosphate of the size secreted by human platelets (approximately 75-mer) acts at two other points in the blood clotting cascade: it accelerates factor V activation and it enhances fibrin clot structure (Smith & Morrissey, Blood, in press). We now compare the ability of RNA and polyphosphate of varying chain lengths to modulate the blood clotting cascade at these three critical points: initiation, factor V activation, and fibrin polymerization. Methods: Polyphosphate was size-fractionated and its procoagulant activities were compared to those of polyinosinic acid, a synthetic singlestranded RNA (ssRNA); polyinosinic acid:polycytidylic acid, a synthetic double-stranded RNA (dsRNA); yeast tRNA or kaolin. Clotting assays were performed using purified fibrinogen, pooled normal plasma, or factor V-deficient plasma to which factor Va was added. Clotting was initiated by CaCl2 (contact pathway), factor Xa, or thrombin. Results: Long-chain polyphosphate (100-mer to 800-mer) triggered the contact pathway with a potency similar to kaolin and was about 30-fold more potent than ssRNA and some 3000- fold more potent than dsRNA or tRNA. Medium-chain polyphosphate (20-mer to 100-mer) and ssRNA both shortened factor Xa clotting times with similar potency, but dsRNA and yeast tRNA were without effect. Replacing plasma factor V with Va blocked the ability of either polyphosphate or ssRNA to shorten factor Xa clotting times, suggesting that both polymers accelerate factor V activation. And finally, when purified fibrinogen was clotted with thrombin, adding either ssRNA or polyphosphate yielded fibrin clots that were about threefold more turbid, indicating that both polymers enhance fibrin clot formation, while dsRNA and yeast tRNA were without effect. (We previously documented that polyphosphate enhances fibrin turbidity by dramatically increasing fibril diameter.) Interestingly, ssRNA significantly shortened the thrombin clotting time of purified fibrinogen, while polyphosphate had no effect on thrombin clotting times. Furthermore, the ability of polyphosphate to enhance fibrin clot structure was calcium-dependent, while ssRNA enhancement of fibrin clotting by thrombin was metal ion-independent. Conclusions: This study shows that RNA modulates critical downstream clotting functions in addition to its previously identified role in triggering the contact pathway. Long-chain polyphosphate (i.e., the size that accumulate in microorganisms, but not the size secreted by platelets) is substantially more potent than RNA in triggering the contact pathway of blood clotting. We therefore propose that polyphosphate may play an important role in host responses to pathogens by triggering the contact pathway. Polyphosphate of the size secreted by platelets had similar potency to ssRNA in accelerating factor V activation. And finally, polyphosphate of the size secreted by platelets had similar potency to RNA in enhancing fibrin clot structure, although the metal ion-dependencies of the two differed, as did their effects on thrombin clotting time. In general, ssRNA was far more potent than dsRNA or tRNA in modulating the blood clotting system.

scholarly journals A Circulating Inhibitor (Anti-AcG) Specific for the Labile Factor-V of the Blood-Clotting Mechanism

Blood ◽  
1958 ◽  
Vol 13 (4) ◽  
pp. 382-397 ◽  
Author(s):  
JOHN H. FERGUSON ◽  
CHARLES L. JOHNSTON ◽  
DORIS A. HOWELL
Keyword(s):  
Blood Clotting ◽  
Factor V ◽  
Blood Clotting System ◽  
Clotting System ◽  
Essential Component ◽  
Test Systems ◽  
Two Phases

Abstract Study of the present hemorrhagic disorder establishes, without doubt, the presence of a powerful specific anti-AcG. The deprivation of AcG due to this inhibitor confirms the view that the labile factor plays a key role in the blood clotting system in at least two phases, namely, (1) during thromboplastin generation, and (2) later, during the conversion of prothrombin to thrombin. AcG is an essential component of many special clotting-test systems.

scholarly journals Polyphosphate enhances fibrin clot structure

Blood ◽  
2008 ◽  
Vol 112 (7) ◽  
pp. 2810-2816 ◽  
Author(s):  
Stephanie A. Smith ◽  
James H. Morrissey
Keyword(s):  
Fibrin Clot ◽  
Factor Xiiia ◽  
Clot Lysis ◽  
Factor V ◽  
Scanning Electron Micrographs ◽  
Dependent Manner ◽  
Dense Granules ◽  
Contact Pathway ◽  
Clot Structure ◽  
Fibrin Clots

Abstract Polyphosphate, a linear polymer of inorganic phosphate, is present in platelet dense granules and is secreted on platelet activation. We recently reported that polyphosphate is a potent hemostatic regulator, serving to activate the contact pathway of blood clotting and accelerate factor V activation. Because polyphosphate did not alter thrombin clotting times, it appeared to exert all its procoagulant actions upstream of thrombin. We now report that polyphosphate enhances fibrin clot structure in a calcium-dependent manner. Fibrin clots formed in the presence of polyphosphate had up to 3-fold higher turbidity, had higher mass-length ratios, and exhibited thicker fibers in scanning electron micrographs. The ability of polyphosphate to enhance fibrin clot turbidity was independent of factor XIIIa activity. When plasmin or a combination of plasminogen and tissue plasminogen activators were included in clotting reactions, fibrin clots formed in the presence of polyphosphate exhibited prolonged clot lysis times. Release of polyphosphate from activated platelets or infectious microorganisms may play an important role in modulating fibrin clot structure and increasing its resistance to fibrinolysis. Polyphosphate may also be useful in enhancing the structure of surgical fibrin sealants.

Dermatan Sulfate and LMW Heparin Enhance the Anticoagulant Action of Activated Protein C

1999 ◽  
Vol 82 (11) ◽  
pp. 1462-1468 ◽  
Author(s):  
José Fernández ◽  
Jari Petäjä ◽  
John Griffin
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Protein C ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor V ◽  
Factor Va ◽  
Anticoagulant Action

SummaryUnfractionated heparin potentiates the anticoagulant action of activated protein C (APC) through several mechanisms, including the recently described enhancement of proteolytic inactivation of factor V. Possible anticoagulant synergism between APC and physiologic glycosaminoglycans, pharmacologic low molecular weight heparins (LMWHs), and other heparin derivatives was studied. Dermatan sulfate showed potent APC-enhancing effect. Commercial LMWHs showed differing abilities to promote APC activity, and the molecular weight of LMWHs correlated with enhancement of APC activity. Degree of sulfation of the glycosaminoglycans influenced APC enhancement. However, because dextran sulfates did not potentiate APC action, the presence of sulfate groups per se on a polysaccharide is not sufficient for APC enhancement. As previously for unfractionated heparin, APC anticoagulant activity was enhanced by glycosaminoglycans when factor V but not factor Va was the substrate. Thus, dermatan sulfate and LMWHs exhibit APC enhancing activity in vitro that could be of physiologic and pharmacologic significance.

Studies on the Pathogenesis of the Generalized Shwartzman Reaction

1964 ◽  
Vol 12 (02) ◽  
pp. 471-483 ◽  
Author(s):  
F Rodríguez-Erdmann
Keyword(s):  
Clotting Factor ◽  
Factor V ◽  
Clotting System ◽  
Platelet Factor ◽  
Trigger Mechanism ◽  
Haemorrhagic Diathesis ◽  
Shwartzman Reaction ◽  
Ca Ions

SummaryThe rôle of the clotting system in the pathogenesis of the generalized Shwartzman reaction (gSr) has been stressed in recent years. The clotting system is activated ubiquitously and as a result of it, fibrin is deposited intravascularly and a haemorrhagic diathesis develops. Evidence is presented herein, that endotoxin does not activate purified prothrombin, nor does endotoxin influence the convertion of prothrombin when it is activated in the presence of purified platelet-factor 3 (or caephalin) purified Ac-G (factor V) and Ca-ions.The trigger mechanism of the gSr also seems to be in the so-called prephase of clotting mechanism. Data are presented, which show that endotoxin activates the Hageman factor in vitro. The importance of this clotting factor and of platelet-factor 3 is discussed. Also the rôle played by the RES and cardiodynamic and vascular components are taken in consideration in the discussion.

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