Evangelina B. Steinbrenner

Tobacco smoke-induced hypercoagulation in human plasma: Role of carbon monoxide

Virtually every disease state associated with chronic or acute thrombosis has had smoking identified as a risk factor. Further, smoking enhances clot strength as assessed by thrombelastography. Critically, carbon monoxide, a product of cigarette smoking, has been demonstrated to enhance plasmatic coagulation in vitro via modulation of a heme associated with fibrinogen. We hypothesized that plasmatic hypercoagulability and formation of carboxyhemefibrinogen (COHF) detected with thrombelastographic methods would be observed after cigarette smoking. Smoking participants (n = 20, two cigarettes consumed within 90 min, average carboxyhemoglobin concentration of 5%) had plasma collected and normal participant (n = 20) plasma was also obtained. Thrombelastographic analyses revealed that plasma obtained from smokers had an 86% greater velocity of clot growth and 65% larger clot strength than normal participant plasma. Forty-five percent of smokers had plasma clot strength that exceeded the 95th percentile of normal participant plasma values; 45% of smoking participants had detectable COHF; and 20% of smoking participants were both hypercoagulable with COHF present. We conclude that smoking induced a hypercoagulable state and COHF formation in an important portion of participants tested. Future investigations of the effects of smoking, plasmatic hypercoagulation and COHF formation are planned in populations with established atherosclerotic/thrombotic disease.

Plasmatic hypercoagulation in patients with breast cancer: Role of heme oxygenase-1

Breast cancer is an important health threat to women worldwide, and is associated with a 9–14% incidence of thrombophilia. Of interest, patients with breast cancer have been noted to have an increase in endogenous carbon monoxide production via upregulation of heme oxygenase-1 activity. Given that it has been demonstrated that carbon monoxide enhances plasmatic coagulation in vitro and in vivo, we sought to determine whether patients with breast cancer had an increase in endogenous carbon monoxide and concurrent plasmatic hypercoagulability. Breast cancer patients who were not smokers scheduled to undergo partial or complete mastectomy (n = 18) had 15 ml of whole blood collected via an indwelling intravenous catheter and anticoagulated with sodium citrate. Whole blood was centrifuged and citrated plasma assessed with a thromboelastometric method to measure coagulation kinetics and the formation of carboxyhemefibrinogen. Breast cancer patients were determined to have an abnormally increased carboxyhemoglobin concentration of 2.5 ± 1.3%, indicative of heme oxygenase-1 upregulation. Breast cancer patient plasma on average clotted 73% more quickly and had 32% stronger thrombus strength than normal individual (n = 30) plasma. Further, 44% of breast cancer patients had plasma clot strength that exceeded the 95% confidence interval value observed in normal individuals, and 75% of this hypercoagulable subgroup had carboxyhemefibrinogen formation. Future investigation of the role played by heme oxygenase-1-derived carbon monoxide in the pathogenesis of breast cancer-related thrombophilia is warranted.

Hemodialysis patients have plasmatic hypercoagulability and decreased fibrinolytic vulnerability: Role of carbon monoxide

Chronic hemodialysis is associated with significant thrombophilia. Of interest, hemodialysis patients have increased carboxyhemoglobin (COHb) and exhaled carbon monoxide (CO), signs of upregulated heme oxygenase (Hmox) activity. Given that CO enhances plasmatic coagulation, we determined whether patients requiring chronic hemodialysis had an increase in endogenous CO, plasmatic hypercoagulability and decreased fibrinolytic vulnerability. Carbon monoxide was determined by noninvasive pulse oximetry measurement of COHb. Blood samples were obtained just before hemodialysis. Thrombelastographic methods to assess plasma coagulation kinetics, fibrinolytic kinetics, and formation of carboxyhemefibrinogen (COHF) were used. Hemodialysis patients (n = 45) had abnormally increased COHb concentrations of 2.2 ± 1.9%, indicative of Hmox upregulation. Coagulation and fibrinolytic parameter normal values were determined with normal individual (n = 30) plasma. Thirty-seven patients of the hemodialysis cohort had COHF formation (82.2%, [67.9%–92.0%]; mean, [95% confidence interval]), and many of this group of patients had abnormally great velocity of clot growth (73.3%, [58.1%–85.4%]) and strength (75.6%, [60.5%–87.1%]). Furthermore, over half of COHF positive patients had a hypofibrinolytic state, evidenced by an abnormally prolonged time to maximum rate of lysis (53.3%, [37.9%–68.6%]) and clot lysis time (64.4%, [48.8%–78.1%]). Carbon monoxide enhanced coagulation and diminished fibrinolytic vulnerability in hemodialysis patients. Future investigation of hemodialysis, CO-related thrombophilia is warranted.

Bariatric patients have plasmatic hypercoagulability and systemic upregulation of heme oxygenase activity.

Morbid obesity is associated with significant thrombophilia. Of interest, adipocytes obtained from obese patients have increased heme oxygenase (Hmox) activity, the endogenous enzyme responsible for carbon monoxide (CO) production. Given that CO enhances plasmatic coagulation, we determined whether morbidly obese patients undergoing bariatric surgery had an increase in endogenous CO and plasmatic hypercoagulability. CO was determined by noninvasive pulse oximetry measurement of carboxyhemoglobin (COHb). A thrombelastographic method to assess plasma coagulation kinetics and formation of carboxyhemefibrinogen (COHF) was utilized. Nonsmoking bariatric patients (n = 20, BMI 47 ± 8 kg/m2, mean ± SD) had abnormally increased COHb concentrations of 2.7 ± 1.9%, indicative of Hmox upregulation. When coagulation kinetics of these bariatric patients were compared with values obtained from normal individuals’ (n = 30) plasma, 70% (95% confidence interval 45.7–88.1%) had abnormally great velocity of clot formation, abnormally large clot strength, and COHF formation. Future investigation of Hmox-derived CO in the pathogenesis of obesity-related thrombophilia is warranted.

Chronic Migraineurs Form Carboxyhemefibrinogen and Iron-Bound Fibrinogen.

Chronic migraine (CM) is a disabling painful condition that is associated with dementia and thrombotic disease. It has been proposed that carbon monoxide (CO) and iron may play a role in CM, and CO and iron are products of the heme oxygenase system which is widespread within the brain. Further, CO and iron enhance plasmatic coagulation in part via a fibrinogen-dependent mechanism. Thus, our goal was to determine whether patients with CM had experienced carboxyhemefibrinogen formation, iron bound fibrinogen formation and plasmatic hypercoagulability. Nonsmokers with CM were recruited after informed, written consent. Blood was collected, anticoagulated with sodium citrate, and then centrifuged with plasma stored at -80ºC. Carboxyhemefibrinogen formation, iron bound fibrinogen formation and coagulation kinetics were determined via thrombelastographic methods. Patient results were compared with laboratory values generated from normal control plasmas. Incidence (95% confidence intervals) of the various parameters was determined using the Clopper-Pearson method. Twenty-six CM patients (24 female) were recruited; they were 46±12 years old. With regard to fibrinogen modification, 88.5% (69.8%-97.6%) of CM patients had formation of carboxyhemefibrinogen, iron bound fibrinogen, or both. With regard to coagulation, 42.3% (23.4%-63.1%) of patients had abnormally decreased time to clot initiation, 80.8% (60.6%-93.4%) had abnormally large velocity of clot formation, and 46.2% (26.6%-66.7%) had abnormally strong clot strength. Patients with CM have a large incidence of carboxyhemefibrinogen and iron bound fibrinogen formation and hypercoagulability. Confirmatory and potential therapeutic clinical trials targeting CO and iron modified hypercoagulation as a source of pain and vascular disease in CM patients are indicated.

Thoracic tumor effects on plasmatic coagulation: role of hemeoxygenase-1.

OBJECTIVES Lung cancer is an important health threat worldwide, and is associated with a 3.8-13.9% incidence of thrombophilia. Of interest, patients with lung tumors have been noted to have an increase in endogenous carbon monoxide production via upregulation of hemeoxygenase-1 activity. Given that it has been demonstrated that carbon monoxide enhances plasmatic coagulation in vitro and in vivo via formation of carboxyhemefibrinogen, we sought to determine if patients with thoracic tumors undergoing lung resection/pneumonectomy had an increase in endogenous carbon monoxide and concurrent plasmatic hypercoagulability. MATERIALS AND METHODS Nonsmoking patients with thoracic tumors (n=19) had preoperative carboxyhemoglobin (a measure of carbon monoxide production) determined, and a thromboelastometric method to assess citrated plasma coagulation kinetics and the formation of carboxyhemefibrinogen was utilized. Thoracic tumor patient coagulation kinetics was compared with normal subject (n=30) plasma samples. RESULTS AND CONCLUSION Patients with thoracic tumors were determined to have an abnormally increased carboxyhemoglobin concentration of 2.1±0.6%, indicative of hemeoxygenase-1 upregulation. It was found that 84% of thoracic tumor patients had plasma clot strength that exceeded the 95% confidence interval value observed in normal subjects, and 44% of this hypercoagulable subgroup had carboxyhemefibrinogen formation. Future investigation of the role played by plasmatic hypercoagulability and hemeoxygenase-1 derived carboxyhemefibrinogen in the pathogenesis of thoracic tumor related thrombophilia is warranted.

Tissue-type plasminogen activator-induced fibrinolysis is enhanced in patients with breast, lung, pancreas and colon cancer:

Although cancer-mediated changes in hemostatic proteins unquestionably promote hypercoagulation, the effects of neoplasia on fibrinolysis in the circulation are less well defined. The goals of the present investigation were to determine if plasma obtained from patients with breast, lung, pancreas and colon cancer was less or more susceptible to lysis by tissue-type plasminogen activator (tPA) compared to plasma obtained from normal individuals. Archived plasma obtained from patients with breast (n = 18), colon/pancreas (n = 27) or lung (n = 19) was compared to normal individual plasma (n = 30) using a thrombelastographic assay that assessed fibrinolytic vulnerability to exogenously added tPA. Plasma samples were activated with tissue factor/celite, had tPA added, and had data collected until clot lysis occurred. Additional, similar samples had potato carboxypeptidase inhibitor added to assess the role played by thrombin-activatable fibrinolysis inhibitor in cancer-modulated fibrinolysis. Rather than inflicting a hypofibrinolytic state, the three groups of cancers demonstrated increased vulnerability to tPA (e.g. decreased time to lysis, increased speed of lysis, decreased clot lysis time). However, hypercoagulation manifested as increased speed of clot formation and strength compensated for enhanced fibrinolytic vulnerability, resulting in a clot residence time that was not different from normal individual thrombi. In sum, enhanced hypercoagulability associated with cancer was in part diminished by enhanced fibrinolytic vulnerability to tPA.

Brain tumors enhance plasmatic coagulation: the role of hemeoxygenase-1.

BACKGROUND:Patients with brain tumors suffer significant thrombotic morbidity and mortality. In addition to increased thrombin generation via tumor release of tissue factor-bearing microparticles and hyperfibrinogenemia, brain tumors and surrounding normal brain likely generate endogenous carbon monoxide (CO) via the hemeoxygenase-1 (HO-1) system. CO has been shown to enhance plasmatic coagulation via formation of carboxyhemefibrinogen (COHF). Thus, our goals in this study were to determine whether patients with brain tumors had increased HO-1 upregulation/CO production, plasmatic hypercoagulability, and formation of COHF. METHODS:Patients with brain tumors (N = 20) undergoing craniotomy had blood collected for determination of carboxyhemoglobin as a marker of HO-1 activity, plasmatic hypercoagulability (defined as clot strength > 95% confidence interval value of normal subject plasma), and COHF formation (determined with a thrombelastograph®-based assay). Plasma obtained from commercially available normal subjects (N = 30) was used for comparison with brain tumor patient samples. RESULTS:Brain tumor patients had carboxyhemoglobin concentrations of 1.5% ± 0.5% (mean ± SD), indicative of HO-1 upregulation. Compared with normal subject plasma, brain tumor patient plasma had significantly (P < 0.0001) greater clot formation velocity (5.2 ± 1.5 vs 9.5 ± 2.3 dynes/cm2/s, respectively) and significantly (P = 0.00016) stronger final clot strength (166 ± 28 vs 230 ± 78 dynes/cm2, respectively). Ten of the brain tumor patients had plasma clot strength that exceeded the 95% confidence interval value observed in normal subjects, and 12 of the brain tumor patients had COHF formation. Five of the brain tumor patients in the hypercoagulable subgroup had COHF formation. Last, 5 of the hypercoagulable patients had primary brain tumors, whereas the other 5 patients had metastatic tumors or an inflammatory mass lesion. CONCLUSIONS:A subset of patients with brain tumors has increased endogenous CO production, plasmatic hypercoagulability, and COHF formation. Future investigation of the role played by HO-1 derived CO in the pathogenesis of brain tumor-associated thrombophilia is warranted.

Colon and pancreas tumors enhance coagulation: role of hemeoxygenase-1.

Colon and pancreatic cancer are associated with significant thrombophilia. Colon and pancreas tumor cells have an increase in hemeoxygenase-1 (HO-1) activity, the endogenous enzyme responsible for carbon monoxide production. Given that carbon monoxide enhances plasmatic coagulation, we determined if patients undergoing resection of colon and pancreatic tumors had an increase in endogenous carbon monoxide and plasmatic hypercoagulability. Patients with colon (n = 17) and pancreatic (n = 10) tumors were studied. Carbon monoxide was determined by the measurement of carboxyhemoglobin (COHb). A thrombelastographic method to assess plasma coagulation kinetics and formation of carboxyhemefibrinogen (COHF) was utilized. Nonsmoking patients with colon and pancreatic tumors had abnormally increased COHb concentrations of 1.4 ± 0.9 and 1.9 ± 0.7%, respectively, indicative of HO-1 upregulation. Coagulation analyses comparing both tumor groups demonstrated no significant differences in any parameter; thus the data were combined for the tumor groups for comparison with 95% confidence interval values obtained from normal individuals (n = 30) plasma. Seventy percent of tumor patients had a velocity of clot formation greater than the 95% confidence interval value of normal individuals, with 53% of this hypercoagulable group also having COHF formation. Further, 67% of tumor patients had clot strength that exceeded the normal 95% confidence interval value, and 56% of this subgroup had COHF formation. Finally, 63% of all tumor patients had COHF formation. Future investigation of HO-1-derived carbon monoxide in the pathogenesis of colon and pancreatic tumor-related thrombophilia is warranted.

Can divergent plasmin-antiplasmin-carbon monoxide interactions in young, healthy tobacco smokers explain the 'smoker's paradox'?

In the setting of acute myocardial infarction, decreases in early/late mortality, reocclusion after thrombolysis, and restenosis rate after percutaneous intervention are lower in smokers – this phenomenon has been designated as the ‘smokers paradox’. These benefits of smoking, however, are abrogated by stent placement. We hypothesized that fibrinolytic vulnerability would change in response to smoking, and that inhaled carbon monoxide may play a role. Smoking patients (n = 20, two cigarettes consumed within 90 min, average carboxyhemoglobin concentration of 5%) had plasma collected and normal individual (n = 20) plasma was also obtained. Thrombelastographic analyses conducted with addition of tissue-type plasminogen activator revealed that with the exception of the rate of thrombus generation, there was little difference in fibrinolytic kinetics between normal and smoking individuals. Addition of exogenous carbon monoxide resulted in diminished fibrinolytic response to the same extent in both groups. Subanalyses demonstrated that the smoking cohort had both hyperfibrinolytic and hypofibrinolytic patients as defined by confidence interval (5–95%) values generated from normal individuals. Addition of carbon monoxide reduced hyperfibrinolytic parameter values by 80% in smokers, whereas only a 17% decrease in hypofibrinolytic values changed. Our investigation suggests that ‘smokers paradox’ involves a marked change in the character of the plasmin–antiplasmin–carbon monoxide interaction. Further investigation will be required to further define the molecular mechanism responsible for the ‘smokers paradox’.