scholarly journals The Involvement of Factors XII & XI in the Platelet Coagulant Activity

1977 ◽  
Author(s):  
J.M. Connellan ◽  
S. Bowden ◽  
P.A. Castaldi
Keyword(s):  
Platelet Rich Plasma ◽  
Factor Xii ◽  
Platelet Activity ◽  
Freezing And Thawing ◽  
Clotting Factors ◽  
Factor Xi ◽  
Platelet Factor ◽  
Coagulant Activity ◽  
Plasma Factor ◽  
Rapid Purification

Platelets provide coagulant activities, other than the Phospholipid Platelet factor 3, which can be released from the platelet. In Platelet Poor Plasma, the initiation of the intrinsic pathway is proposed to proceed via factors Xll & XI as well as 2 other factors, Prekallekrein and Kininogen. The role of these factors in the initiation of coagulation in Platelet Rich Plasma is under investigation. Walsh, (Br.J. Haemat. 22. 393, 1972) and Muntz (Haemostasis in press 1977), have shown that platelets devoid of factor Xll will still produce a coagulant activity when Platelet Rich Plasma is incubated with collagen. However, we have recently shown that this reaction requires the presence of factor XI. Factor XII has been purified by the method of Saito (Circ.Res.34,641,1974) , and factor XI has been purified by the method of Connellan, Castaldi and Muntz. (Haemostasis, in press 1977).Specific antibodies were developed in rabbits and the IgG from the antiserum was coupled to Sepharose. These factors could be extracted from plasma by the immobilized antibodies and could then be eluted by the use of alkali or guanidine HCL, thus resulting in a rapid purification of these factors.Platelets were washed free of loosely bound clotting factors and provided significant amounts of coagulant activity when ruptured by either freezing and thawing or by glycerol treatment. Platelets from a factor XI deficient patient also provided this activity. The platelet activity was extracted by passage through both Sepharose-heparin column and Sepharose-anti-factor XI column;the activity appeared to be identical to plasma factor XI.

Plasma Factor XIII and Platelet Factor XIII in Hyperlipaemia

1976 ◽  
Vol 36 (03) ◽  
pp. 542-550 ◽  
Author(s):  
Mircea P. Cucuianu ◽  
K Miloszewski ◽  
D Porutiu ◽  
M. S Losowsky
Keyword(s):  
Factor Xiii ◽  
Significant Contribution ◽  
Quantitative Assay ◽  
Factor Xii ◽  
Platelet Factor ◽  
Type Iv ◽  
Plasma Factor ◽  
Hepatic Synthesis

SummaryPlasma factor XIII activity measured by a quantitative assay was found to be significantly higher in hypertriglyceridaemic patients (type IV and combined hyperlipoproteinaemia), as compared to normolipaemic controls. No such elevation in plasma factor XIII activity was found in patients with type IIa hyperlipaemia. Plasma pseudocholinesterase was found to parallel the elevated factor XIII activity in hypertriglyceridaemic subjects.In contrast, platelet factor XIII activity was not raised in hyperlipaemic subjects, and plasma factor XIII was found to be normal in a normolipaemic subject with throm-bocythaemia.It was concluded that there is no significant contribution from platelets to plasma factor XIII activity, and that the observed increase in plasma factor XII in hypertriglyceridaemia results from enhanced hepatic synthesis of the enzyme.

scholarly journals Fibronectin modulates formation of PF4/heparin complexes and is a potential factor for reducing risk of developing HIT

Blood ◽  
2019 ◽  
Vol 133 (9) ◽  
pp. 978-989 ◽  
Author(s):  
Krystin Krauel ◽  
Patricia Preuße ◽  
Theodore E. Warkentin ◽  
Catja Trabhardt ◽  
Sven Brandt ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Enzyme Linked Immunosorbent Assay ◽  
Heparin Binding ◽  
Plasma Fibronectin ◽  
Platelet Factor ◽  
Washed Platelet ◽  
Plasma Factor ◽  
Heparin Complexes ◽  
Patient Groups

Abstract Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating anti–platelet factor 4 (PF4)/heparin antibodies. Platelet activation assays that use “washed” platelets are more sensitive for detecting HIT antibodies than platelet-rich plasma (PRP)–based assays. Moreover, heparin-exposed patients vary considerably with respect to the risk of PF4/heparin immunization and, among antibody-positive patients, the risk of subsequent “breakthrough” of clinical HIT with manifestation of thrombocytopenia. We used washed platelets and PRP, standard laboratory HIT tests, and physicochemical methods to identify a plasma factor interfering with PF4/heparin complexes and anti-PF4/heparin antibody–platelet interaction, thus explaining differences in functional assays. To investigate a modulating risk for PF4/heparin immunization and breakthrough of HIT, we also tested 89 plasmas from 2 serosurveillance trials. Fibronectin levels were measured in 4 patient groups exhibiting different degrees of heparin-dependent immunization and expression of HIT. The heat-labile plasma protein, fibronectin, inhibited PF4 binding to platelets in a dose-dependent fashion, particularly in washed (vs PRP) systems. Fibronectin also inhibited PF4/heparin binding to platelets, anti-PF4/heparin antibody binding to PF4/heparin complexes, and anti-PF4/heparin antibody–induced platelet activation as a result of PF4/heparin complex disruption. In addition, plasma fibronectin levels increased progressively among the following 4 patient groups: enzyme-linked immunosorbent assay (ELISA)+/serotonin-release assay (SRA)+/HIT+ < ELISA+/SRA+/HIT− ∼ ELISA+/SRA−/HIT− < ELISA−/SRA−/HIT−. Altogether, these findings suggest that fibronectin interferes with PF4/heparin complex formation and anti-PF4/heparin antibody–induced platelet activation. Reduced fibronectin levels in washed platelet assays help to explain the greater sensitivity of washed platelet (vs PRP) assays for HIT. More importantly, lower plasma fibronectin levels could represent a risk factor for PF4/heparin immunization and clinical breakthrough of HIT.

scholarly journals Factor V activity of platelets: evidence for an activated factor V molecule and for a platelet activator

Blood ◽  
1977 ◽  
Vol 49 (5) ◽  
pp. 819-834 ◽  
Author(s):  
B Osterud ◽  
SI Rapaport ◽  
KK Lavine
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Factor V ◽  
Freezing And Thawing ◽  
Early Step ◽  
Platelet Factor ◽  
Plasma Factor ◽  
Native Factor

Abstract This study was prompted by the observation that fresh platelet suspensions--prepared by gel filtration or albumin density gradient centrifugation--possessed only minimal factor V activity, whereas frozen-and-thawed platelet suspensions possessed striking factor V activity. Results of experiments with fresh suspensions suggested that unaltered platelets did not bind plasma factor V. The factor V activity of frozen-and-thawed platelet suspensions was markedly diminished after exposure to a factor V antibody, was not activated by thrombin, and was not associated with an increase in factor V antigen over that found in fresh platelet suspensions. Consequently, disruption by freezing and thawing must have resulted in the appearance of small amounts of an activated factor V molecule in platelet suspensions. Disrupted platelets were shown to activate native factor V, but an interaction between a platelet activator and traces of native factor V in fresh suspensions could not be demonstrated to account for the full activity of frozen-and-thawed suspensions. Apparently, therefore, platelets also contained an activated factor V molecule. Adding collagen, but not adenosine 5′-diphosphate to fresh platelet suspensions increased their factor V activity. Release of an activated platelet factor V molecule after exposure to collagen could represent a physiologically significant early step in hemostasis.

scholarly journals Properties of sulfatides in factor-XII-dependent contact activation

Blood ◽  
1982 ◽  
Vol 59 (1) ◽  
pp. 69-75 ◽  
Author(s):  
G Tans ◽  
JH Griffin
Keyword(s):  
Normal Values ◽  
Limited Proteolysis ◽  
Factor Xii ◽  
Contact Activation ◽  
Amidolytic Activity ◽  
Prolonged Incubation ◽  
Coagulant Activity ◽  
Plasma Factor ◽  
Normal Human ◽  
Kallikrein Activity

Abstract Incubation of normal human plasma with low amounts of sulfatides resulted in the initiation of intrinsic coagulation and the appearance of kallikrein activity. The optimal initiation of procoagulant and kallikrein amidolytic activity was dependent on the presence of factor XII, high molecular weight kininogen, and prekallikrein. Since the activated partial thromboplastin clotting times in prekallikrein- deficient plasma approach normal values upon prolonged incubation with kaolin, this phenomenon of autocorrection was studied and found to be even more pronounced in the presence of sulfatides. Autocorrection was essentially completed in 5 min in the presence of sulfatides, whereas a preincubation of 15–20 min was required in the presence of kaolin. The limited proteolysis of 125I-factor XII in plasma during incubation with activating material or during clotting was determined. Cleavage of factor XII was more rapid and more extensive in the presence of sulfatides than in the presence of kaolin. In prekallikrein-deficient plasma, factor XII cleavage was completed within 5 min in the presence of sulfatides and within 15 min in the presence of kaolin. Thus, the appearance of factor-XII-dependent coagulant activity correlates with the limited proteolysis of factor XII when normal or prekallikrein- deficient plasma is activated by sulfatides or kaolin.

scholarly journals Tissue-Specific Expression of Functional Platelet Factor XI Is Independent of Plasma Factor XI Expression

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3800-3807 ◽  
Author(s):  
Chang-jun Hu ◽  
Frank A. Baglia ◽  
David C.B. Mills ◽  
Barbara A. Konkle ◽  
Peter N. Walsh
Keyword(s):  
Binding Sites ◽  
Factor Xi ◽  
Specific Expression ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Tissue Specific ◽  
Factor Xi Deficiency ◽  
Plasma Factor ◽  
Granule Membrane ◽  
Granule Secretion

Abstract Platelet factor XI is an alternatively spliced product of the factor XI gene expressed specifically within megakaryocytes and platelets as an approximately 1.9-kb mRNA transcript (compared with ∼2.1 kb in liver cells) lacking exon V. Flow cytometry with an affinity-purified factor XI antibody, with PAC1 antibody (to the GPIIb/IIIa complex on activated platelets), and with S12 antibody (to P-selectin, an α-granule membrane protein expressed on the platelet surface during secretion) on platelets activated with ADP, thrombin, thrombin receptor peptide (SFLLRN amide), or collagen at various concentrations exposed platelet factor XI and PAC1 antibody binding in parallel. Unactivated platelets expressed approximately 40% of total platelet factor XI but no PAC1 binding sites. Enhanced membrane exposure of platelet factor XI is independent of α-granule secretion, because ADP and collagen exposed platelet factor XI but no S12 binding sites. Platelets from four patients with plasma factor XI deficiency (<0.04 U/mL) had normal constitutive and activation-dependent expression of platelet factor XI. Well-washed platelets from normal and from factor XI-deficient donors incubated with low concentrations of thrombin (0.05 to 0.1 U/mL) corrected the clotting defect observed with factor XI-deficient plasma. Thus, functionally active platelet factor XI is differentially expressed on platelet membranes in a tissue-specific manner both constitutively and in a concentration-dependent fashion by various agonists in the absence of detectable plasma factor XI.

Effect of Heparin on the Activation of Factor XI by Fibrin-bound Thrombin

1996 ◽  
Vol 76 (03) ◽  
pp. 347-353 ◽  
Author(s):  
Peter A Kr von dem Borne ◽  
Joost C M Meijers ◽  
Bonno N Bouma
Keyword(s):  
Antithrombin Iii ◽  
Anticoagulant Activity ◽  
Factor Xii ◽  
Binding Studies ◽  
Factor Xi ◽  
Limited Effectiveness ◽  
Coagulant Activity ◽  
Fibrin Monomers ◽  
Activation Capacity

SummaryFibrin-bound thrombin is protected from inactivation by antithrombin III, while its coagulant potential is retained. In the presence of heparin, ternary complexes between thrombin, fibrin and heparin are formed. In these complexes the coagulant activity of thrombin is retained, whereas the anticoagulant activity of fibrin-bound heparin is neutralized. The limited effectiveness of heparin in the prevention of both venous thrombosis and coronary reocclusion is probably related to the protective effect of fibrin on the inactivation of thrombin by antithrombin III. Recently, it has been shown that factor XI can be activated by thrombin, resulting in the generation of additional thrombin via the intrinsic pathway. This additional thrombin is capable of stabilizing the clot by protecting it from fibrinolysis. We studied the effect of heparin on the activation of factor XI by fibrin-bound thrombin. First, we used fibrin monomers coupled to Sepharose to which thrombin and unfractionated heparin (UFH) were bound. Factor XI activation by thrombin was the same in the presence of fibrin-Sepharose or control-Sepha-rose. The addition of heparin (0.1 U/ml) resulted in a 91 and 15-fold enhancement in the presence of control-Sepharose and fibrin-Sepharose, respectively. Next, we added complexes of heparin, thrombin and fibrin monomer to factor XII and XI double-deficient plasma in the presence or absence of a reconstituting amount of factor XI. In the presence of factor XI, additional fibrin formation was observed indicating that factor XI activation by thrombin in complex with fibrin and heparin can take place in plasma. We then studied the effect of other heparin-like anticoagulants on the thrombin-mediated factor XI activation. UFH enhanced thrombin-mediated factor XI activation 68-fold, LMWH (low molecular weight heparin, Fragmin) 12-fold, danaparoid (Orgaran) 3-fold, while the pentasaccharide ORG 31540 did not result in an enhancement. Binding studies of these anticoagulants to fibrin-Sepharose showed that LMWH bound with approximately the same affinity as UFH, while danaparoid and the pentasaccharide did not bind to fibrin.We conclude that fibrin-bound thrombin is capable of factor XI activation. Furthermore, heparin bound in a complex with fibrin can act as a cofactor for this activation. This factor XI activation capacity may play a role in the limited effectiveness of heparin. Provided that thrombin-mediated factor XI activation plays an important role in vivo, danaparoid and especially the pentasaccharide may be better anticoagulants than UFH and LMWH.

scholarly journals Activation of Hageman Factor (HF, Factor XII) by Sephadex-Ellagic Acid Mixtures

1977 ◽  
Author(s):  
O. D. Ratnoff ◽  
H. Saito
Keyword(s):  
Ellagic Acid ◽  
Partial Thromboplastin Time ◽  
Factor Xii ◽  
Intrinsic Pathway ◽  
Clotting Factors ◽  
Factor Xi ◽  
Molecular Changes ◽  
Hageman Factor ◽  
Procoagulant Effect ◽  
Fletcher Factor

In the intrinsic pathway of clotting, PTA (Factor XI) is activated by activated HF. Whether activation of HF requires the presence of prekallikrein (Fletcher factor) and high M. W.(HMW) kininogen (Fitzgerald factor), or whether these agents act after activation of HF is disputed. Purified HF activates PTA in the absence of prekallikrein, but this may reflect molecular changes in HF during purification.Sephadex G10 or G15 was mixed with ellagic acid and freed of excess ellagic acid by washing. A mixture of normal plasma, or plasma deficient in HF, prekallikrein, HMW kininogen or PTA, and Sephadex-ellagic acid was centrifuged at 31000 xg. Each supernatant plasma except that deficient in HF shortened the abnormally long partial thromboplastin time (PTT) of HF-deficient plasma. Plasma simultaneously depleted of HMW kininogen, prekallikrein and PTA also shortened the PTT of HF-deficient plasma and of plasma depleted of HF and prekallikrein (albeit minimal clotting times were reached more slowly in the latter case) but had virtually no procoagulant effect upon the PTT of plasma depleted of HF and HMW kin inogen.Thus, exposure of HF in plasma to Sephadex-ellagic acid generated a clot-promoting form of HF in the absence of other clotting factors, but its full expression requited the presence of HMW kininogen.

scholarly journals Factor IX is activated in vivo by the tissue factor mechanism

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 731-736 ◽  
Author(s):  
KA Bauer ◽  
BL Kass ◽  
H ten Cate ◽  
JJ Hawiger ◽  
RD Rosenberg
Keyword(s):  
Tissue Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor Xii ◽  
Factor Xi ◽  
Factor Xi Deficiency ◽  
Factor Ixa ◽  
Coagulant Activity ◽  
The Mean

Abstract Despite significant progress in elucidating the biochemistry of the hemostatic mechanism, the process of blood coagulation in vivo remains poorly understood. Factor IX is a vitamin K-dependent glycoprotein that can be activated by factor XIa or the factor VII-tissue factor complex in vitro. To investigate the role of these two pathways in factor IX activation in humans, we have developed a sensitive procedure for quantifying the peptide that is liberated with the generation of factor IXa. The antibody population used for the immunoassay was raised in rabbits and chromatographed on a factor IX-agarose immunoadsorbent to obtain antibody populations with minimal intrinsic reactivity toward factor IX. We determined that the mean level of the factor IX activation peptide (FIXP) in normal individuals under the age of 40 years was 203 pmol/L and that levels increased significantly with advancing age. The mean concentration of FIXP was markedly reduced to 22.7 pmol/L in nine patients with hereditary factor VII deficiency (factor VII coagulant activity less than 7%) but was not significantly different from normal controls in nine subjects with factor XI deficiency (factor XI coagulant activity less than 8%). These data indicate that factor IXa generation in vivo results mainly from the activity of the tissue factor mechanism rather than the contact system (factor XII, prekallikrein, high molecular-weight kininogen, factor XI). Our results may also help to explain the absence of a bleeding diathesis in many patients with deficiencies of the contact factors of coagulation.

The Effect Of Plasmin On Factor XIII (Fibrin Stabilizing Factor)

1981 ◽  
Author(s):  
D M Rider ◽  
J M McDonagh
Keyword(s):  
Factor Xiii ◽  
Activate Factor ◽  
Polyacrylamide Gels ◽  
Clotting Factors ◽  
Human Fibrinogen ◽  
Platelet Factor ◽  
Plasma Factor ◽  
Before And After ◽  
A Subunit ◽  
Almost All

The action of plasmin on several blood clotting factors has been studied; however, controversy exists concerning the effect of plasmin on factor XIII. Factor XIII was purified from plasma and platelets and then exposed to plasmin for up to 6 hours. Plasmin to factor XIII ratios ranged from 0.03-0.1 casein units plasmin per mg factor XIII. These plasmin levels exhibited strong proteolytic activities against B-casein and purified human fibrinogen Following incubation of factor XIII (activated and unactivated) with plasmin the mixtures were electrophoresed on 7% SDS-polyacrylamide gels. The factor XIII preparations were assayed for 14C-putrescine incorporating activity before and after exposure to plasmin. Platelet factor XIII was,labeled With 125Iodine and lableled a subunit (activated and unactivated) was exposed to plalmin for up to 2 hours. These mixtures were electrophoresed on 12.5% Urea-SDS Polyacrylamide gels and a radioactivity profile was determined for each gel.Following extensive exposure to Plasmin the relative molecular weights of the factor XIII subunits (a, a* and b)remained constant and almost all (90-100%) of the 14C-put-rescine incorporating activity was recovered. The radio-activity profiles of the gels of 125I-labeled platelet factor XIII were identical before and after incubation with plasmin. Plasmin did not activate factor XIII in the assay system nor did factor XIII inactivate plasmin by crosslinking it. These experiments indicate that plasmin does not activate or degrade factor XIII and that the b subunit of plasma factor XIII plays no role in protecting the a subunit from the action of plasmin.

scholarly journals Amidolytic assay of human factor XI in plasma: comparison with a coagulant assay and a new rapid radioimmunoassay

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 42-50 ◽  
Author(s):  
CF Scott ◽  
D Sinha ◽  
FS Seaman ◽  
PN Walsh ◽  
RW Colman
Keyword(s):  
Trypsin Inhibitor ◽  
Coefficient Of Variation ◽  
Protein A ◽  
Poor Correlation ◽  
Factor Xii ◽  
Staphylococcal Protein A ◽  
Contact Activation ◽  
Factor Xi ◽  
Plasma Factor ◽  
The Mean

Abstract The traditional coagulant assay for plasma factor XI suffers from a relatively high coefficient of variation, the need for rare congenitally deficient plasma, and a poor correlation between precision and sensitivity. We have developed a simple functional amidolytic assay for factor XI in plasma using the chromogenic substrate PyrGlu-Pro-Arg- p-nitroanilide (S-2366). After inactivation of alpha 1-antitrypsin, CI inhibitor, and other plasma protease inhibitors with CHCI3, plasma was incubated with kaolin, in the absence of added calcium, which limited the enzymes formed to those dependent on contact activation. Soybean trypsin inhibitor was used to minimize the action of kallikrein on the substrate. Once the reaction was complete, corn trypsin inhibitor was used to inactive factor XIIa, the enzyme generated by exposure of plasma to negatively charged surfaces, which had activated the factor XI. The assay is highly specific for factor XI, since plasma totally deficient in that zymogen yielded only 1%-3% of the enzymatic activity in normal plasma under identical conditions. The requirements for complete conversion of factor XI to XIa in plasma within 60 min were, respectively, factor XII, 0.6 U/ml, and high molecular weight kininogen, 0.2 U/ml. Prekallikrein was not an absolute requirement for complete activation but did accelerate the reaction. The intraassay coefficient of variation was 3.4%, and the mean of 35 normal plasmas was 1.00 U +/- 0.24 SD. In addition, a new rapid radioimmunoassay was devised using staphylococcal protein A as the precipitating agent for a complex of factor XI antigen with monospecific rabbit antibody. The mean was 1.01 U +/- 0.30 SD. The correlation coefficients for amidolytic versus coagulant and amidolytic versus radioimmunoassay were r = 0.95 for the former and 0.96 for the latter. Thus, a simple, accurate amidolytic assay and a radioimmunoassay have been devised for measuring factor XI in plasma that correlate well with the coagulant activity of factor XI, as determined in our laboratory.

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