Lipoprotein-associated coagulation inhibitor (LACI) is a cofactor for heparin: synergistic anticoagulant action between LACI and sulfated polysaccharides

Abstract Lipoprotein-associated coagulation inhibitor (LACI) is a plasma-derived protein that inhibits tissue factor (TF)/factor VIIa-induced coagulation in a factor Xa-dependent manner. The roles of endogenous plasma LACI and exogenously added LACI and heparin, in the regulation of coagulation, initiated via the intrinsic and extrinsic pathways, were studied using the activated partial thromboplastin time (APTT) and the modified prothrombin time (PT) assays, respectively. Both LACI- depleted plasma and normal plasma have identical APTTs and similar prolongations of the APTT in response to heparin; both are fully anticoagulated (arbitrarily defined as clotting times of greater than 1 hour) at similar concentrations of heparin. These results indicate that heparin is an effective anticoagulant when coagulation is initiated by the intrinsic pathway and that endogenous LACI is not significantly involved in the regulation of this pathway. The PT of normal plasma is only marginally longer than that of LACI-depleted plasma in the absence of heparin, suggesting that endogenous plasma LACI has a very limited capacity to inhibit TF-induced clotting. However, in the presence of heparin, the PTs of LACI-depleted plasma and normal plasma are different. Prolongation of the PT occurred only moderately and linearly with increasing concentrations of heparin in LACI-depleted plasma. In contrast, normal plasma showed a greater extent of PT prolongation in response to heparin and the plasma became fully anticoagulated at a certain threshold concentration of heparin. These results suggest that LACI serves as a cofactor for heparin and thus greatly enhances the inhibition of TF-induced coagulation. LACI-depleted plasma was supplemented with purified recombinant LACI and/or heparin and the effects on TF-induced clotting were studied. A combination of LACI and heparin greatly enhanced anticoagulation compared with LACI or heparin alone. Many sulfated polysaccharides were also found to enhance the LACI-dependent inhibition of TF-induced clotting. By weight, the relative potencies of these compounds are: low molecular weight heparin (mean Mr, 5,100) greater than unfractionated heparin greater than low molecular weight heparin (mean Mr, 3,700) greater than pentosan polysulfate greater than dermatan sulfate greater than dextran sulfate greater than heparan sulfate. Based on the above results, it is concluded that LACI is a cofactor for heparin in the inhibition of TF- induced clotting and that LACI and sulfated polysaccharides act synergistically in whole plasma.</jats:p

Lipoprotein-associated coagulation inhibitor (LACI) is a cofactor for heparin: synergistic anticoagulant action between LACI and sulfated polysaccharides

Lipoprotein-associated coagulation inhibitor (LACI) is a plasma-derived protein that inhibits tissue factor (TF)/factor VIIa-induced coagulation in a factor Xa-dependent manner. The roles of endogenous plasma LACI and exogenously added LACI and heparin, in the regulation of coagulation, initiated via the intrinsic and extrinsic pathways, were studied using the activated partial thromboplastin time (APTT) and the modified prothrombin time (PT) assays, respectively. Both LACI- depleted plasma and normal plasma have identical APTTs and similar prolongations of the APTT in response to heparin; both are fully anticoagulated (arbitrarily defined as clotting times of greater than 1 hour) at similar concentrations of heparin. These results indicate that heparin is an effective anticoagulant when coagulation is initiated by the intrinsic pathway and that endogenous LACI is not significantly involved in the regulation of this pathway. The PT of normal plasma is only marginally longer than that of LACI-depleted plasma in the absence of heparin, suggesting that endogenous plasma LACI has a very limited capacity to inhibit TF-induced clotting. However, in the presence of heparin, the PTs of LACI-depleted plasma and normal plasma are different. Prolongation of the PT occurred only moderately and linearly with increasing concentrations of heparin in LACI-depleted plasma. In contrast, normal plasma showed a greater extent of PT prolongation in response to heparin and the plasma became fully anticoagulated at a certain threshold concentration of heparin. These results suggest that LACI serves as a cofactor for heparin and thus greatly enhances the inhibition of TF-induced coagulation. LACI-depleted plasma was supplemented with purified recombinant LACI and/or heparin and the effects on TF-induced clotting were studied. A combination of LACI and heparin greatly enhanced anticoagulation compared with LACI or heparin alone. Many sulfated polysaccharides were also found to enhance the LACI-dependent inhibition of TF-induced clotting. By weight, the relative potencies of these compounds are: low molecular weight heparin (mean Mr, 5,100) greater than unfractionated heparin greater than low molecular weight heparin (mean Mr, 3,700) greater than pentosan polysulfate greater than dermatan sulfate greater than dextran sulfate greater than heparan sulfate. Based on the above results, it is concluded that LACI is a cofactor for heparin in the inhibition of TF- induced clotting and that LACI and sulfated polysaccharides act synergistically in whole plasma.</jats:p

Immunoradiometric quantitation of tissue plasminogen activator-related antigen in human plasma: crypticity phenomenon and relationship to plasma fibrinolysis

A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L- arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA- Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above.</jats:p

Initiation and regulation of fibrinolysis in human plasma at the plasminogen activator level

Abstract The initiation and regulation of fibrinolysis has been studied by reconstitution of fibrinolytic activity in human plasma in vitro. Depletion of tissue plasminogen activator (tPA) antigen by immunoadsorption of human plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous fibrinolytic activity determined by lysis of a thrombin-induced clot. Addition of physiological concentrations of purified tPA to tPA-depleted plasma restores fibrinolytic activity as a function of the length of time between tPA addition and clotting. Addition of free tPA to tPA-depleted plasma followed by immediate clotting results in a high rate of fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in unclotted plasma is accompanied by decreased partitioning of tPA antigen into fibrin after clotting and is kinetically correlated with the formation of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel electrophoresis and fibrin-agar zymography. These results suggest that free tPA is susceptible to complexation by the plasma inhibitor in the absence of a clot. Fibrin formation renders tPA relatively inaccessible to inhibition. The tPA antigen isolated from stored plasma consists mainly of 100 kDa activity in SDS-gel electrophoresis and zymography, indicating that the tPA complex is resistant to dissociation by SDS. Upon rezymography of the sliced gel, only a 60 kDa tPA activity is found, suggesting that the activity at 100 kDa is at least partly due to free tPA dissociated from the complex during the first zymography. Conversion of tPA complex to enzymatically active free tPA also occurs with brief SDS exposure followed by incubation in the presence of excess Triton X-100 or by hydroxylamine treatment. These results reconcile the apparent discrepancy of the 100 kDA inhibitor-tPA complex manifesting plasminogen activation activity during zymography. The plasma tPA- inhibitor complex is precipitated strongly by antisera against plasminogen activator inhibitors (PAIs) of human Hep G2 hepatoma and HT- 1080 fibrosarcoma cells and weakly by antiserum against bovine aortic endothelial cell PAI but not by antiserum against a placental PAI (PAI- 2) suggesting that the plasma inhibitor is immunologically related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the above findings, a simple model for the initiation and regulation of plasma fibrinolysis at the PA level has been formulated.</jats:p

Initiation and regulation of fibrinolysis in human plasma at the plasminogen activator level

The initiation and regulation of fibrinolysis has been studied by reconstitution of fibrinolytic activity in human plasma in vitro. Depletion of tissue plasminogen activator (tPA) antigen by immunoadsorption of human plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous fibrinolytic activity determined by lysis of a thrombin-induced clot. Addition of physiological concentrations of purified tPA to tPA-depleted plasma restores fibrinolytic activity as a function of the length of time between tPA addition and clotting. Addition of free tPA to tPA-depleted plasma followed by immediate clotting results in a high rate of fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in unclotted plasma is accompanied by decreased partitioning of tPA antigen into fibrin after clotting and is kinetically correlated with the formation of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel electrophoresis and fibrin-agar zymography. These results suggest that free tPA is susceptible to complexation by the plasma inhibitor in the absence of a clot. Fibrin formation renders tPA relatively inaccessible to inhibition. The tPA antigen isolated from stored plasma consists mainly of 100 kDa activity in SDS-gel electrophoresis and zymography, indicating that the tPA complex is resistant to dissociation by SDS. Upon rezymography of the sliced gel, only a 60 kDa tPA activity is found, suggesting that the activity at 100 kDa is at least partly due to free tPA dissociated from the complex during the first zymography. Conversion of tPA complex to enzymatically active free tPA also occurs with brief SDS exposure followed by incubation in the presence of excess Triton X-100 or by hydroxylamine treatment. These results reconcile the apparent discrepancy of the 100 kDA inhibitor-tPA complex manifesting plasminogen activation activity during zymography. The plasma tPA- inhibitor complex is precipitated strongly by antisera against plasminogen activator inhibitors (PAIs) of human Hep G2 hepatoma and HT- 1080 fibrosarcoma cells and weakly by antiserum against bovine aortic endothelial cell PAI but not by antiserum against a placental PAI (PAI- 2) suggesting that the plasma inhibitor is immunologically related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the above findings, a simple model for the initiation and regulation of plasma fibrinolysis at the PA level has been formulated.</jats:p

Immunoradiometric quantitation of tissue plasminogen activator-related antigen in human plasma: crypticity phenomenon and relationship to plasma fibrinolysis

Abstract A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L- arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA- Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above.</jats:p