Coagulation, Fibrinolytic, and Inhibitory Proteins in Acute Myocardial Infarction and Angina Pectoris N.D. VAZIRI,M.D., SEANC. KENNEDY,M.D., DANIELKENNEDY,M.D., EMMAGONZALES,M.D., Irvine, California
BACKGROUND AND METHODi The role of thrombus formation in the pathogenesis of acute myocardial infarction (AMI) and unstable angina pectmis has been well established. However, comprehensive and systematic studies of the blood coagulation, fibrinolytic, and inhibitory proteins are not available in patients with these conditions. Fourteen patients with AMI, 10 patients with angina pectoris, and 32 normal volunteers were studied Plasma antigen concentrations and/or activities of high-molecular-weight lrininogen (HMWE), fibrinogen, fibronectin, plasminogen, D-dimer, tissue plasminogen activator (t-PA), ark-antiplasmin, a2-macroglobulin, al-antitrypsh, protein C, total and free protein S, antithrombin III (AT-III), von Willebrand factor (vWF), factors (F) XII, XI, IX, VIII, VII, X, V, II, and XIII, and plasma antiplasmin activity were measured using appropriate functional or immunologic assays. RESULTS The AMI group showed a significant reduction in F XII activity, F XII activity-toconcentration ratio, and HMWE concentration. In addition, the AMI patients exhibited a significant elevation of plasma F XI activity, F IX concentration, and F IX activity, and vWF, fibriuogen, Ddimer, and t-PA concentrations. This was associated with significant reductions in F V, F II, and AT-III activity-to-concentration ratio. Many of the changes observed in AMI patients were also present in patients with angina pectoris. Furthermore, the latter group exhibited an elevation of F VIII activity, cu~macroglobulin activity, and q-antitrypsin concentration and a significant reduction of antiplasmin activity despite a normal cwrantiplasmin concentration
From
the Department
of Medicine,
University
of California,
Irvine.
Requestsfor reprintsshould be addressed to N. D. Vaziri, M.D., Department of Medicine, UCI Medical Center, 101 The City Drive, Building 53 Manuscript
submitted
November
12. 1991, and accepted
in revised
CONCLUSIONS:Theobservedreduction ofthe plasma F XII activity-to-antigen concentration ratio combined with a reduced HMWK concentration suggests intrinsic pathway activation, while the elevation of the D-dimer concentration indicates thrombin generation and fibrin formation and degradation in the AM1 group. The latter changes were also present in patients with angina pectoris. Both AMI and augina groups showed several other abnormalities of the coagulation, fibrinolytic, and inhibitory systems. The results suggest the presence of a prothrombotic state associated with the activation of coagulation and fibrinolytic systems iu patients with acute myocardkl ischemia or infarction.
everal angiographic, angioscopic, and postmorS tem investigations have clearly demonstrated the role of thrombus formation in the pathogenesis of acute myocardial infarction (AMI) and unstable angina [l-5]. This viewpoint is further supported by several indirect observations: (1) Use of antiplatelet agents has been shown to significantly reduce the risk of AM1 in susceptible individuals [6,7]; (2) An elevated plasma fibrinogen concentration has been identified as an independent risk factor for ischemic cardiovascular disease [8]; and (3) Thrombolytic therapy has been shown to decrease the extent of acute infarction and contribute to preservation of myocardial function [9]. In addition, generation of thrombin and products of platelet activation have been thought to contribute to the formation and evolution of atherosclerotic lesions by promoting smooth muscle cell and fibroblast proliferation [19-121. It is, therefore, clear that blood coagulation plays a major role in the development and outcome of coronary atherosclerosis. However, available data on the coagulation and related systems in patients with ischemic heart disease and in those progressing to AM1 are limited. We undertook the present study to evaluate the status of the entire blood coagulation cascade, fibrinolytic system, and inhibitory proteins in a group of patients with AM1 and those with unstable angina.
December 1992 The American Journal of Medicine
Volume 93
651
COAGULATION
SYSTEM
IN ACUTE
MYOCARDIAL
l9CtlEMlA
/ VAZIRI ET AL
The resultswerecomparedwith thoseobtainedin a congenitalhuman F VII-deficient plasma,congenigroup of normal controls. tal human F X-deficient plasma,agedhuman F Vdeficient plasma, and F II-deficient substrateprePATIENTS, MATERIALS,AND METHODS pared from aged human serum and barium Patients sulfate-adsorbedbovine plasma. Fourteen consecutive patients (10 men and 4 The antisera against human high-molecularwomen,aged48to 80years)presentingto the hospi- weight kininogen (HMWK) and F XII were purtal with AMI were included in the study. Ten con- chasedfrom ICN Inc. (Lisle, IL). Antiserum against secutive patients (8 men and 2 women, aged51 to human F IX was purchasedfrom American Bio79)presentingwith clinical and electrocardiograph- products (Parsippany,NJ), and antiserato human ic evidenceof myocardial &hernia (anginapecto- F II, protein C, protein S, cYz-antiplasmin, fibronecris) without acuteinfarction werealsostudied. The tin, and F XIIIa-subunit werepurchasedfrom Caldiagnosisof AM1 wassuspectedon clinical grounds biochem Inc. (La Jolla, CA). Coagulationassayed and confiied by the presenceof appropriatenew- referenceplasmapurchasedfrom HelenaLaboratoonset electrocardiographicchanges and elevated ries (Beaumont,TX) servedas the normal pooled creatine kinase MB isoenxyme.Eleven of the 14 plasma standard. patients in the AM1 group showedQ-waveinfarcSuppliesfor determination of antigen concentration whereasthe remaining 3 had non-Q-wavein- tions of the following proteins werepurchasedfrom farction. Presentation to the hospital occurred American Bioproducts (Parsippany,NJ): von Wilwithin 8 hours of the onset of chest pain. lebrand factor (vWF), F VII, F X, D-dimer, and Exclusion criteria included discernible liver dis- tissue plasminogenactivator (t-PA). Supplies for ease,primary coagulopathy,nephrotic syndrome, antithrombin III (AT-III) antigen concentration, and use of oral contraceptive medications, anti- plasminogenconcentration, and cws-macroglobulin platelet agents,and other drugsknown to affect the concentrationassayswere purchasedfrom Helena blood coagulation system. Laboratories(Beaumont,TX). Fibrinogen concentration was determinedusing a kit purchasedfrom Control Group Sigma Laboratories (St. Louis, MO). Supplies for Thirty-two normal volunteers (23 men and 9 protein C amidolytic activity determination were women, aged 20 to 49 years)were included in the purchasedfrom American Diagnostics(Greenwich, study. None of the participants had a history of CT), and thosefor AT-III activity were purchased cardiacdisease,hypertension,liver disease,tobacco from Helena Laboratories (Beaumont, TX). smoking, kidney disease,or coagulopathy. They had a normal prothrombin time and a normal par- Functional Assays tial thromboplastin time, and had not used oral Procoagulant activities of factors XII, XI, IX, contraceptivemedicationsor other drugs known to and VIII were determined in a one-stageclotting affect the blood coagulationsystem. assayusing a modification of partial thromboplastin time [13], and plasma coagulant activities of Blood Samples factors II, V, VII, and X weredetermined in a twoFree-flowing venousblood sampleswere collect- stageclotting assayusing a modification of Quick ed at the time of presentation to the emergency time [14]. department (within 8 hours after the onsetof chest Antiplasmin activity wasdetermined by an amipain) in plastic tubes containing 3.8% sodium ci- dolytic method employing plasmin excessand a trate solution (blood/citrate = 9/l volume) using chromogenicsubstratein which the amount of plasplastic syringes.Sampleswere immediately centri- min activity inhibited is proportional to the amount fuged at 4,OOOXg for 20 minutes. The plasma was of antiplasmin present in the test sample. separatedand stored in multiple small ahquotaat ai-Antitrypsin activity was measuredusing ex-7OOCuntil assayed. cesstrypsin, which is inhibited by cul-antitrypsin. The remaining amount of free trypsin is deterMaterials mined by measuringthe amidolytic activity of reThe following factor (F)-deficient plasma sub- sidual trypsin on the chromogenicsubstrate. AT-III activity was measured using excess strateswere purchasedfrom Dade DiagnosticsInc. (Miami, FL): congenital human F XII-deficient thrombin, addedto heparinizedplasma sample,to plasma, congenital bovine F XI-deficient plasma, react with antithrombin-heparin complexes. The human F IX-deficient substrate.preparedfrom true residual thrombin is then allowed to react with a hemophilia B plasma,human F VIII-deficient sub- synthetic chromogenic substrate, p-nitroaniline, strate prepared from true hemophilia A plasma, which is measuredphotometrically. 652
December
1992
The American
Journal
of Medlclne
Volume
93
COAGULATION SYSTEM IN ACUTE MYOCARDIAL ISCHEMIA / VAZIRI ET AL TABLE I Plasma Procoagulant Activities (Act) and/or Antigen (Ag) Concentrations(Cone)of High-Molecular-Weight Kininogen (HMWK), von Willebrand Factor (vWF),and Factors(F) XII, XI, IX, and VIII in the Study Groups
Ac(u$e=mgardiai
infarction
Normal control (n = 32) Unstable angina (n =
10)
74 f 4.2
BACKGROUND AND METHODi The role of thrombus formation in the pathogenesis of acute myocardial infarction (AMI) and unstable angina pectmis has been well established. However, comprehensive and systematic studies of the blood coagulation, fibrinolytic, and inhibitory proteins are not available in patients with these conditions. Fourteen patients with AMI, 10 patients with angina pectoris, and 32 normal volunteers were studied Plasma antigen concentrations and/or activities of high-molecular-weight lrininogen (HMWE), fibrinogen, fibronectin, plasminogen, D-dimer, tissue plasminogen activator (t-PA), ark-antiplasmin, a2-macroglobulin, al-antitrypsh, protein C, total and free protein S, antithrombin III (AT-III), von Willebrand factor (vWF), factors (F) XII, XI, IX, VIII, VII, X, V, II, and XIII, and plasma antiplasmin activity were measured using appropriate functional or immunologic assays. RESULTS The AMI group showed a significant reduction in F XII activity, F XII activity-toconcentration ratio, and HMWE concentration. In addition, the AMI patients exhibited a significant elevation of plasma F XI activity, F IX concentration, and F IX activity, and vWF, fibriuogen, Ddimer, and t-PA concentrations. This was associated with significant reductions in F V, F II, and AT-III activity-to-concentration ratio. Many of the changes observed in AMI patients were also present in patients with angina pectoris. Furthermore, the latter group exhibited an elevation of F VIII activity, cu~macroglobulin activity, and q-antitrypsin concentration and a significant reduction of antiplasmin activity despite a normal cwrantiplasmin concentration
From
the Department
of Medicine,
University
of California,
Irvine.
Requestsfor reprintsshould be addressed to N. D. Vaziri, M.D., Department of Medicine, UCI Medical Center, 101 The City Drive, Building 53 Manuscript
submitted
November
12. 1991, and accepted
in revised
CONCLUSIONS:Theobservedreduction ofthe plasma F XII activity-to-antigen concentration ratio combined with a reduced HMWK concentration suggests intrinsic pathway activation, while the elevation of the D-dimer concentration indicates thrombin generation and fibrin formation and degradation in the AM1 group. The latter changes were also present in patients with angina pectoris. Both AMI and augina groups showed several other abnormalities of the coagulation, fibrinolytic, and inhibitory systems. The results suggest the presence of a prothrombotic state associated with the activation of coagulation and fibrinolytic systems iu patients with acute myocardkl ischemia or infarction.
everal angiographic, angioscopic, and postmorS tem investigations have clearly demonstrated the role of thrombus formation in the pathogenesis of acute myocardial infarction (AMI) and unstable angina [l-5]. This viewpoint is further supported by several indirect observations: (1) Use of antiplatelet agents has been shown to significantly reduce the risk of AM1 in susceptible individuals [6,7]; (2) An elevated plasma fibrinogen concentration has been identified as an independent risk factor for ischemic cardiovascular disease [8]; and (3) Thrombolytic therapy has been shown to decrease the extent of acute infarction and contribute to preservation of myocardial function [9]. In addition, generation of thrombin and products of platelet activation have been thought to contribute to the formation and evolution of atherosclerotic lesions by promoting smooth muscle cell and fibroblast proliferation [19-121. It is, therefore, clear that blood coagulation plays a major role in the development and outcome of coronary atherosclerosis. However, available data on the coagulation and related systems in patients with ischemic heart disease and in those progressing to AM1 are limited. We undertook the present study to evaluate the status of the entire blood coagulation cascade, fibrinolytic system, and inhibitory proteins in a group of patients with AM1 and those with unstable angina.
December 1992 The American Journal of Medicine
Volume 93
651
COAGULATION
SYSTEM
IN ACUTE
MYOCARDIAL
l9CtlEMlA
/ VAZIRI ET AL
The resultswerecomparedwith thoseobtainedin a congenitalhuman F VII-deficient plasma,congenigroup of normal controls. tal human F X-deficient plasma,agedhuman F Vdeficient plasma, and F II-deficient substrateprePATIENTS, MATERIALS,AND METHODS pared from aged human serum and barium Patients sulfate-adsorbedbovine plasma. Fourteen consecutive patients (10 men and 4 The antisera against human high-molecularwomen,aged48to 80years)presentingto the hospi- weight kininogen (HMWK) and F XII were purtal with AMI were included in the study. Ten con- chasedfrom ICN Inc. (Lisle, IL). Antiserum against secutive patients (8 men and 2 women, aged51 to human F IX was purchasedfrom American Bio79)presentingwith clinical and electrocardiograph- products (Parsippany,NJ), and antiserato human ic evidenceof myocardial &hernia (anginapecto- F II, protein C, protein S, cYz-antiplasmin, fibronecris) without acuteinfarction werealsostudied. The tin, and F XIIIa-subunit werepurchasedfrom Caldiagnosisof AM1 wassuspectedon clinical grounds biochem Inc. (La Jolla, CA). Coagulationassayed and confiied by the presenceof appropriatenew- referenceplasmapurchasedfrom HelenaLaboratoonset electrocardiographicchanges and elevated ries (Beaumont,TX) servedas the normal pooled creatine kinase MB isoenxyme.Eleven of the 14 plasma standard. patients in the AM1 group showedQ-waveinfarcSuppliesfor determination of antigen concentration whereasthe remaining 3 had non-Q-wavein- tions of the following proteins werepurchasedfrom farction. Presentation to the hospital occurred American Bioproducts (Parsippany,NJ): von Wilwithin 8 hours of the onset of chest pain. lebrand factor (vWF), F VII, F X, D-dimer, and Exclusion criteria included discernible liver dis- tissue plasminogenactivator (t-PA). Supplies for ease,primary coagulopathy,nephrotic syndrome, antithrombin III (AT-III) antigen concentration, and use of oral contraceptive medications, anti- plasminogenconcentration, and cws-macroglobulin platelet agents,and other drugsknown to affect the concentrationassayswere purchasedfrom Helena blood coagulation system. Laboratories(Beaumont,TX). Fibrinogen concentration was determinedusing a kit purchasedfrom Control Group Sigma Laboratories (St. Louis, MO). Supplies for Thirty-two normal volunteers (23 men and 9 protein C amidolytic activity determination were women, aged 20 to 49 years)were included in the purchasedfrom American Diagnostics(Greenwich, study. None of the participants had a history of CT), and thosefor AT-III activity were purchased cardiacdisease,hypertension,liver disease,tobacco from Helena Laboratories (Beaumont, TX). smoking, kidney disease,or coagulopathy. They had a normal prothrombin time and a normal par- Functional Assays tial thromboplastin time, and had not used oral Procoagulant activities of factors XII, XI, IX, contraceptivemedicationsor other drugs known to and VIII were determined in a one-stageclotting affect the blood coagulationsystem. assayusing a modification of partial thromboplastin time [13], and plasma coagulant activities of Blood Samples factors II, V, VII, and X weredetermined in a twoFree-flowing venousblood sampleswere collect- stageclotting assayusing a modification of Quick ed at the time of presentation to the emergency time [14]. department (within 8 hours after the onsetof chest Antiplasmin activity wasdetermined by an amipain) in plastic tubes containing 3.8% sodium ci- dolytic method employing plasmin excessand a trate solution (blood/citrate = 9/l volume) using chromogenicsubstratein which the amount of plasplastic syringes.Sampleswere immediately centri- min activity inhibited is proportional to the amount fuged at 4,OOOXg for 20 minutes. The plasma was of antiplasmin present in the test sample. separatedand stored in multiple small ahquotaat ai-Antitrypsin activity was measuredusing ex-7OOCuntil assayed. cesstrypsin, which is inhibited by cul-antitrypsin. The remaining amount of free trypsin is deterMaterials mined by measuringthe amidolytic activity of reThe following factor (F)-deficient plasma sub- sidual trypsin on the chromogenicsubstrate. AT-III activity was measured using excess strateswere purchasedfrom Dade DiagnosticsInc. (Miami, FL): congenital human F XII-deficient thrombin, addedto heparinizedplasma sample,to plasma, congenital bovine F XI-deficient plasma, react with antithrombin-heparin complexes. The human F IX-deficient substrate.preparedfrom true residual thrombin is then allowed to react with a hemophilia B plasma,human F VIII-deficient sub- synthetic chromogenic substrate, p-nitroaniline, strate prepared from true hemophilia A plasma, which is measuredphotometrically. 652
December
1992
The American
Journal
of Medlclne
Volume
93
COAGULATION SYSTEM IN ACUTE MYOCARDIAL ISCHEMIA / VAZIRI ET AL TABLE I Plasma Procoagulant Activities (Act) and/or Antigen (Ag) Concentrations(Cone)of High-Molecular-Weight Kininogen (HMWK), von Willebrand Factor (vWF),and Factors(F) XII, XI, IX, and VIII in the Study Groups
Ac(u$e=mgardiai
infarction
Normal control (n = 32) Unstable angina (n =
10)
74 f 4.2
