Hemostasis and its Regulation

2016 ◽  
Author(s):  
Lawrence L K Leung
Keyword(s):  
Endothelial Cells ◽  
Blood Coagulation ◽  
Protein C ◽  
Formation Control ◽  
Blood Clot ◽  
Protein S ◽  
Clot Formation ◽  
Tissue Type ◽  
Control Mechanisms ◽  
Clotting Cascade

Hemostasis, the process of blood clot formation, is a coordinated series of responses to vessel injury. It requires complex interactions between platelets, the clotting cascade, blood flow and shear, endothelial cells, and fibrinolysis. This review covers platelet plug formation, clotting cascade, initiation and propagation of blood clot formation, control mechanisms, overview of blood coagulation, blood coagulation as part of the host defense system, heterogeneity of endothelial cells and vascular bed–specific hemostasis, platelet production and thrombopoietin, and coagulation tests and their use. Figures show activated platelets, platelet aggregation, the classic and revised view of the clotting cascade, the inhibition of thrombin by antithrombin, the protein C/protein S pathway, the synergism between nitric oxide (NO) and prostacyclin (PGI2), tissue-type plasminogen activator, the transformation of fibrinogen to fibrin, activated protein C (APC) and carboxypeptidase B-2 (CPB-2) at the site of vascular injury, and an algorithm detailing the exposure of tissue factor at a vascular wound that initiates the clotting cascade. The table lists natural antithrombotic mechanisms of endothelial cells. This review contains 10 highly rendered figures, 1 table, and 45 references.

scholarly journals The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1189-1192 ◽  
Author(s):  
NJ de Fouw ◽  
F Haverkate ◽  
RM Bertina ◽  
J Koopman ◽  
A van Wijngaarden ◽  
...  
Keyword(s):  
Whole Blood ◽  
Protein C ◽  
Degradation Products ◽  
Blood Clot ◽  
Activated Protein C ◽  
Protein S ◽  
Tissue Type ◽  
Clot Lysis ◽  
Fibrin Degradation Products

Abstract The effect of purified human activated protein C (APC) and protein S on fibrinolysis was studied by using an in vitro blood clot lysis technique. Blood clots were formed from citrated blood (supplemented with 125I-fibrinogen) by adding thrombin and Ca2+-ions; lysis of the clots was achieved by adding tissue-type plasminogen activator. The release of labeled fibrin degradation products from the clots into the supernatant was followed in time. We clearly demonstrated that APC accelerates whole blood clot lysis in vitro. The effect of APC was completely quenched by antiprotein C IgG, pretreatment of APC with diisopropylfluorophosphate, and preincubation of the blood with antiprotein S IgG. This demonstrates that both the active site of APC and the presence of the cofactor, protein S, are essential for the expression of the profibrinolytic properties. At present, the substrate of APC involved in the regulation of fibrinolysis is not yet known. Analysis of the radiolabeled fibrin degradation products demonstrated that APC had no effect on the fibrin cross-linking capacity of factor XIII.

scholarly journals The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1189-1192
Author(s):  
NJ de Fouw ◽  
F Haverkate ◽  
RM Bertina ◽  
J Koopman ◽  
A van Wijngaarden ◽  
...  
Keyword(s):  
Whole Blood ◽  
Protein C ◽  
Degradation Products ◽  
Blood Clot ◽  
Activated Protein C ◽  
Protein S ◽  
Tissue Type ◽  
Clot Lysis ◽  
Fibrin Degradation Products

The effect of purified human activated protein C (APC) and protein S on fibrinolysis was studied by using an in vitro blood clot lysis technique. Blood clots were formed from citrated blood (supplemented with 125I-fibrinogen) by adding thrombin and Ca2+-ions; lysis of the clots was achieved by adding tissue-type plasminogen activator. The release of labeled fibrin degradation products from the clots into the supernatant was followed in time. We clearly demonstrated that APC accelerates whole blood clot lysis in vitro. The effect of APC was completely quenched by antiprotein C IgG, pretreatment of APC with diisopropylfluorophosphate, and preincubation of the blood with antiprotein S IgG. This demonstrates that both the active site of APC and the presence of the cofactor, protein S, are essential for the expression of the profibrinolytic properties. At present, the substrate of APC involved in the regulation of fibrinolysis is not yet known. Analysis of the radiolabeled fibrin degradation products demonstrated that APC had no effect on the fibrin cross-linking capacity of factor XIII.

Activated Protein C Increases Fibrin Clot Lysis by Neutralization of Plasminogen Activator Inhibitor No Evidence for a Cofactor Role of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 328-333 ◽  
Author(s):  
N J de Fouw ◽  
Y F de Jong ◽  
F Haverkate ◽  
R M Bertina
Keyword(s):  
Plasminogen Activator ◽  
Protein C ◽  
Blood Platelets ◽  
Plasminogen Activator Inhibitor ◽  
Protein S ◽  
Tissue Type ◽  
Clot Lysis ◽  
Activator Inhibitor ◽  
Pai 1

summaryThe effect of purified human activated protein G (APC) on fibrinolysis was studied using a clot iysis system consisting of purified glu-plasminogen, tissue-type plasminogen activator, plasminogen activator inhibitor (released from endothelial cells or blood platelets), fibrinogen, 125T-fibrinogen and thrombin. All proteins were of human origin.In this system APC could increase fibrinolysis in a dose dependent way, without affecting fibrin formation or fibrin crosslinking. However, this profibrinolytic effect of APC could only be observed when plasminogen activator inhibitor (PAI-l) was present. The effect of APC was completely quenched by pretreatment of APC with anti-protein C IgG or di-isopropylfluorophosphate. Addition of the cofactors of APC:protein S, Ca2+-ions and phospholipid-alone or in combination did not enhance the profibrinolytic effect of APC. These observations indicate that human APC can accelerate in vitro clot lysis by the inactivation of PAI-1 activity. However, the neutralization of PAI-1 by APC is independent of the presence or absence of protein S, phospholipid and Ca2+-ions.

scholarly journals Participation of endothelial cells in the protein C-protein S anticoagulant pathway: the synthesis and release of protein S.

1986 ◽  
Vol 102 (5) ◽  
pp. 1971-1978 ◽  
Author(s):  
D Stern ◽  
J Brett ◽  
K Harris ◽  
P Nawroth
Keyword(s):  
Endothelial Cells ◽  
Vessel Wall ◽  
Protein C ◽  
Protein S ◽  
Calcium Flux ◽  
Ionophore A23187 ◽  
Intracellular Protein ◽  
Major Band ◽  
C Protein ◽  
Morphological Studies

The protein C-protein S anticoagulant pathway is closely linked to the endothelium. In this paper the synthesis and release of the vitamin K-dependent coagulation factor protein S is demonstrated. Western blotting, after SDS PAGE of Triton X-100 extracts of bovine aortic endothelial cells grown in serum-free medium, demonstrated the presence of protein S. A single major band was observed at Mr approximately 75,000, closely migrating with protein S purified from plasma absent from cells treated with cycloheximide. Metabolic labeling of endothelial cells with [35S]methionine confirmed de novo synthesis of protein S. Using a radioimmunoassay, endothelium was found to release 180 fmol/10(5) cells per 24 h and contain 44 fmol/10(5) cells of protein S antigen. Protein S released from endothelium was functionally active and could promote activated protein C-mediated factor Va inactivation on the endothelial cell surface. Warfarin decreased secretion of protein S antigen by greater than 90% and increased intracellular accumulation by almost twofold. Morphological studies demonstrated intracellular protein S was in the Golgi complex, concentrated at the trans face, rough endoplasmic reticulum, lysosomes, and in vesicles at the periphery. In contrast, protein S was not found in vascular fibroblasts or smooth muscle cells. A pool of intracellular protein S could be released rapidly by the calcium ionophore A23187 (5 microM). This effect was dependent on the presence of calcium in the culture medium and could be blocked by LaCl3, which suggests that cytosolic calcium flux may be responsible for protein S release. These results demonstrate that endothelial cells, but not the subendothelial cells of the vessel wall, can synthesize and release protein S, which indicates a new mechanism by which the inner lining of the vessel wall can contribute to the prevention of thrombotic events.

scholarly journals Modulation of endothelial cell hemostatic properties by tumor necrosis factor.

1986 ◽  
Vol 163 (3) ◽  
pp. 740-745 ◽  
Author(s):  
P P Nawroth ◽  
D M Stern
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tumor Necrosis Factor ◽  
Cell Surface ◽  
Protein C ◽  
Tumor Necrosis ◽  
Protein S ◽  
Active Role ◽  
Procoagulant Activity ◽  
Necrosis Factor

Tumor necrosis factor/cachectin (TNF) is a mediator of the septic state, which involves diffuse abnormalities of coagulation throughout the vasculature. Since previous studies have shown that endothelial cells can play an active role in coagulation, we wished to determine whether TNF could modulate endothelial cell hemostatic properties. Incubation of purified recombinant TNF with cultured endothelial cells resulted in a time- and dose-dependent acquisition of tissue factor procoagulant activity. Concomitant with enhanced procoagulant activity, TNF also suppressed endothelial cell cofactor activity for the anticoagulant protein C pathway; both thrombin-mediated protein C activation and formation of functional activated protein C-protein S complex on the cell surface were considerably attenuated. Comparable concentrations of TNF (half-maximal affect at approximately 50 pM) and incubation times (half-maximal affect by 4 h after addition to cultures) were required for each of these changes in endothelial cell coagulant properties. This unidirectional shift in cell surface hemostatic properties favoring promotion of clot formation indicates that, in addition to leukocyte procoagulants, endothelium can potentially be instrumental in the pathogenesis of the thrombotic state associated with inflammatory and malignant disorders.

scholarly journals The Interaction of Osteoblasts With Bone-Implant Materials: 1. The Effect of Physicochemical Surface Properties of Implant Materials

2011 ◽  
pp. 95-111 ◽  
Author(s):  
D. KUBIES ◽  
L. HIMMLOVÁ ◽  
T. RIEDEL ◽  
E. CHÁNOVÁ ◽  
K. BALÍK ◽  
...  
Keyword(s):  
Surface Roughness ◽  
Free Energy ◽  
Blood Coagulation ◽  
Surface Free Energy ◽  
Blood Clot ◽  
Surface Treatments ◽  
Clot Formation ◽  
Osteoblast Proliferation ◽  
Human Osteoblasts ◽  
Implant Materials

This comparative study of various surface treatments of commercially available implant materials is intended as guidance for orientation among particular surface treatment methods in term of the cell reaction of normal human osteoblasts and blood coagulation. The influence of physicochemical surface parameters such as roughness, surface free energy and wettability on the response of human osteoblasts in the immediate vicinity of implants and on the blood coagulation was studied. The osteoblast proliferation was monitored and the expression of tissue mediators (TNF-α, IL-8, MMP-1, bone alkaline phosphatase, VCAM-1, TGF-β) was evaluated after the cell cultivation onto a wide range of commercially available materials (titanium and Ti6Al4V alloy with various surface treatments, CrCoMo alloy, zirconium oxide ceramics, polyethylene and carbon/carbon composite). The formation of a blood clot was investigated on the samples immersed in a freshly drawn whole rabbit blood using scanning electron microscope. The surfaces with an increased osteoblast proliferation exhibited particularly higher surface roughness (here Ra > 3.5 µm) followed by a high polar part of the surface free energy whereas the effect of wettability played a minor role. The surface roughness was also the main factor regulating the blood coagulation. The blood clot formation analysis showed a rapid coagulum formation on the rough titanium-based surfaces. The titanium with an etching treatment was considered as the most suitable candidate for healing into the bone tissue due to high osteoblast proliferation, the highest production of osteogenesis markers and low production of inflammatory cytokines and due to the most intensive blood clot formation.

Recombinant Thrombomodulin and Activated Protein C in the Treatment of Disseminated Intravascular Coagulation

1999 ◽  
Vol 82 (08) ◽  
pp. 718-721 ◽  
Author(s):  
Ikuro Maruyama
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Disseminated Intravascular Coagulation ◽  
Protein C ◽  
Activated Protein C ◽  
Coagulation Cascade ◽  
Regulation System ◽  
Luminal Surface ◽  
Intravascular Coagulation

IntroductionThe blood coagulation cascade is regulated by the luminal surface of the endothelial cell lining.1 Endothelial cells synthesize tissue factor pathway inhibitor (TFPI), which, in part, binds to the cell surface glycosaminoglycans and inhibits factors Xa, VIIa, and tissue factor.2 Endothelial cells also produce and exhibit thrombomodulin (TM) on their luminal surface.3 TM is a kind of thrombin receptor that forms a 1:1 complex with thrombin. In this complex, thrombin activates protein C (PC) more than 1,000-fold more than thrombin alone. TM then loses its procoagulant activities, which include fibrinogen clotting, activation of factors V and VIII, and platelet activation. Thus, TM converts thrombin from a procoagulant protease to an anticoagulant. Pathologic states, such as an endothelial injury or perturbation or continuous rapid coagulation cascade activation, overcomes the endothelial regulating activity, resulting in the development of intravascular coagulation and the induction of disseminated intravascular coagulation (DIC). Theoretically, then, supplementing soluble TM or activated PC (APC) to reconstitute the endothelial coagulation regulation system in the circulation and regulate pathologically-activated blood coagulation could be beneficial. In this chapter, application of soluble TM and APC in the treatment of DIC is reviewed.

STIMULATION OF FIBRINOLYSIS BY ACTIVATED PROTEIN C (APC)

1987 ◽  
Author(s):  
N J de Four ◽  
R M Bertina ◽  
F Havgrkate
Keyword(s):  
Complex Formation ◽  
Whole Blood ◽  
Blood Platelets ◽  
Blood Clot ◽  
Protein S ◽  
Clot Formation ◽  
Clot Lysis ◽  
Minor Role ◽  
Human Proteins ◽  
Pai 1

In 1960 Mammen and Seegers reported the discovery of a new protein (autoprothrombin II-A, APC) with both anticoagulant and profibrinolytic activity. They found that APC accelerated clot lysis in vitro and proposed that this was due to a reduction of plasmin - inhibitory activity. Many years later Comp et al (J Clin Inv 68: 1221) reported that the infusion of APC into dogs resulted in an increase in circulating plasminogen activator activity. This observation stimulated more extensive studies of the profibrinolytic effects of APC.In our laboratories we have studied the effect of human APC on clot lysis both in whole blood (human) and in a system of purified human proteins. In these systems 125I-labelled fibrinogen was incorporated in a clot formed after the addition of Jombin (complete clot formation within 5 min) and the subsequent lysis of this clot was followed by measuring the release of I-labelled fibrin degradation products (FDP) into the supernatant. Human t-PA was added to the system to achieve complete lysis of the clot within a few hours.When APC was added to citrated whole blood before clot formation, it was found to accelerate clot lysis in a dose dependent way. This effeg| was specific for APC and dependent on an intact active site, on the presence of protein S (the protein cofactor of APC) and Ca . The presence of APC did not influence the composition of the FDP formed, as analysed by means of SDS-polyacry-1 amide gel electroforesis, and its effect was found to be independent of the presence or absence of a.-antiplasmin.Subsequently we developped a clot lysis system using the purified human proteins of the fibrinolytic system: fibrinogen, FXIII, t-PA, PAI-1 (from human endothelial cells), glu-plasminogen and a -antiplasmin. In this system clot lysis was dependent on the concentrations of plasminogen, -antiplasmin, t-PA and PAI-1, but independent on the thrombin concentration and the presence or absence of phospholipids (purified from human brain). In the absence of PAI-1, no effect of APC on clot lysis was observed. However, in the presence of PAI-1, APC accelerated clot lysis. This effect was independent of the presence or absence of phospholipids and/or protein S and could be explained by the observation that APC can form a complex with PAI-1 (~ 95 kd) and under certain conditions even can convert active PAI-1 (~ 46 kd) into an inactive degradation product (~ 42 kd). However, complex formation is relatively slow anti high PAI-1 concentrations are needed to observe the reaction. The addition of protein S or phospholipids in the presence of Ca did not stimulate complex formation. Therefore, it seems highly unlikely that neutralization of PAI-1 by APC is responsible for the profibrinolytic effect of APC in the whole blood clot lysis.A completely different explanation for the profibrinolytic effect of APC was suggested by the observation that the addition of blood-platelets to the system of purified fibrinolytic components introduced a dependence of the clot lysis rate on the thrombin concentration (decrease in clot lysis at increasing thrombin concentration). This finding opened the possibility that APC stimulated fibrinolysis by reducing the effective thrombin concentration. Subsequent experiments using the whole blood clot lysis system revealed that in the presence of anti-FX antibodies clot lysis was no longer accelerated by APC, while the actual rate of clot lysis depended on the concentration of thrombin added.We like to propose, that in a blood clot lysis system APC most likely accelerates fibrinolysis by reducing the effective thrombin concentration; if at all, neutralization of PAI-1 may play only a minor role.

Platelet Factor 4 Mediates Activated Protein C Resistance by Impairment of Protein S Cofactor Enhancement

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 20-20
Author(s):  
Roger JS Preston ◽  
Jennifer A Johnson ◽  
Fionnuala Ni Ainle ◽  
Shona Harmon ◽  
Owen P. Smith ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Endothelial Barrier ◽  
Platelet Factor ◽  
Barrier Permeability ◽  
Gla Domain ◽  
Anti Inflammatory

Abstract Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C to significantly enhance activated protein C (APC) generation by the thrombin-thrombomodulin complex on the surface of endothelial cells. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. The functional consequences of the interaction between the APC Gla domain and PF4 in relation to APC anticoagulant, anti-inflammatory and anti-apoptotic functions have not previously been fully defined. In a tissue factor-initiated thrombin generation assay, APC impaired thrombin generation as previously described. However PF4 inhibited APC anticoagulant activity in a concentration-dependent manner (IC50 for PF4 inhibition of APC anticoagulant function, 11μg/ml). In contrast, addition of two other cationic polypeptides protamine and polybrene, both significantly enhanced APC anticoagulant activity in plasma. To elucidate the mechanism through which PF4 inhibits APC anticoagulant activity, we utilized a phospholipid-dependent FVa proteolysis time course assay. In the absence of protein S, PF4 had no effect upon FVa proteolysis by APC, indicating that PF4 does not influence the ability of APC to interact with either anionic phospholipids or FVa. However, in the presence of protein S, PF4 significantly inhibited APC-mediated FVa proteolysis (3–5 fold). Collectively, these findings demonstrate that in addition to enhancing APC generation, PF4 also significantly attenuates APC anticoagulant activity in plasma by impairing critical protein S cofactor enhancement of FVa proteolysis, and suggest that PF4 contributes to the poorly-understood APC resistance phenotype associated with activated platelets. APC bound to the endothelial cell protein C receptor (EPCR) via its Gla domain can activate PAR-1 on endothelial cells, triggering complex intracellular signaling that result in anti-inflammatory and anti-apoptotic cellular responses. To ascertain whether PF4 interaction with the protein C/APC Gla domain might impair APC-EPCR-PAR-1 cytoprotective signaling, APC protection against thrombin-induced endothelial barrier permeability and staurosporine-induced apoptosis in the presence of PF4 was determined. APC significantly attenuated thrombin-induced endothelial cell barrier permeability, as expected. PF4 alone (up to 1μM) had no independent effect upon endothelial barrier permeability, and did not protect against thrombin-mediated increased permeability. In contrast to its inhibition of APC anticoagulant activity, PF4 did not significantly inhibit the endothelial barrier protective properties of APC. To determine whether PF4 might interfere with APC-mediated cytoprotection, staurosporine-induced apoptosis in EAhy926 cells was assessed by RT-PCR quantification of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) gene expression. Pre-treatment of EAhy926 cells with APC decreased the Bax/Bcl-2 ratio close to that determined for untreated EAhy926 cells. PF4 alone, or in combination with APC, had no effect upon apoptosis-related gene expression as determined by alteration of Bax/Bcl-2 expression ratios in response to staurosporine. In summary, PF4 inhibits APC anticoagulant function via inhibition of essential protein S cofactor enhancement in plasma, whilst retaining EPCR/PAR-1 mediated cytoprotective signalling on endothelial cells. This provides a rationale for how PF4 can exert prothrombotic effects in vivo, but also mediate enhanced APC generation on the surface of endothelial cells to induce both anti-inflammatory and anti-apoptotic events. Based on these observations, we propose that PF4 acts as a critical regulator of APC generation in vivo, but also targets APC towards cytoprotective, rather than anticoagulant functions at sites of vascular injury with concurrent platelet activation.

DETECTION AND EFFECTS OF THROMBOMODULIN ACTIVITY IN CRUDE THROMBOPLASTIN PREPARATIONS FROM PLACENTA AND LUNG

1987 ◽  
Author(s):  
M L Wiesel ◽  
R Spaethe ◽  
J-M Freyssinet ◽  
T Tran ◽  
H-J Kolde ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vitamin K ◽  
Human Placenta ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Coagulation System ◽  
Factor X ◽  
Coagulant Activity ◽  
The Stability

The activation of protein C (PC) by thrombin requires the presence of an endothelial membrane cofactor, thrombomodulin (TM). Activated PC (APC) exerts its anticoagulant activity by degrading factors (F) Va and Villa in the presence of phospholipids and of a vitamin K-dependent cofactor, protein S. Tissue factor (TF) is the essential cofactor of factor Vll/VIIain the activation of factor X. TF is synthetized by several cell lines including endothelial cells. Using a specific TM assay, up to 0.85 units of TM activity could be detected in commercial thromboplastin (TP) preparations from human placenta or rabbit or porcine lung, when the amount of TP was adjusted to contain 1 unit of TF activity. Preparations from brain contained very low amounts, if any, of this activity (< 0.02 TM units). In order to evaluate the effects of the presence of TM activity in some TP preparations, the stability of F V and VIII activities was examined after activation of the coagulation system by these TP in various plasmas. PC deficient plasmas, plasmas lacking F V, VIII or IX and immunoadsorbed PC deficient plasma supplemented with purified human PC (5 Ug/ml) were used. After activation with placenta or lung TP, F V and VIII activities were markedly reduced ( ∼ 90 % reduction) in normal and hemophiliac plasmas, whereas they remained high after activation with brain TP. F V and VIII activities were preserved in protein C deficient plasma after activation by all TP preparations. The same decrease of F V and VIII activities was observed after activation of immunoadsorbed PC deficient plasma supplemented with purified PC with placenta or lung TP only. Preincubation of TP from human placenta with antibodies to human TM raised in laying hens abolished the capacity of this preparation to destroy F V activity of PC containing plasmas. These results establish the presence of TM activity in crude thromboplastin preparations from placenta or from lung. Surprisingly, this anti-coagulant activity seems to be absent from brain. TM from placenta or lung extracts is responsible for the degradation of F V and VIII.

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