BACKGROUND: Intracerebral hemorrhage (ICH) of undetermined etiology occurs infrequently in young and middle-aged adults. We hypothesized that slight decreases in coagulation factors and formation of less compact fibrin clots prone to faster lysis predispose to this type of ICH.
METHODS: We recruited 44 consecutive patients aged <50 years following ICH of unknown cause at least 3 months since the event. Subjects free of ICH (n = 47) matched for age, sex, BMI, and hypertension served as the control group. We assessed plasma fibrin clot permeability, turbidity and fibrinolytic capacity, along with thrombin generation, coagulation factors (F) II, FV, FVII, FVIII, FIX, FX, FXI, antithrombin, and fibrinolysis proteins.
RESULTS: ICH patients (median age 41 years, 45.5 % women) had 8.4 % lower FII (p = 0.0001) and 10.1 % lower FVII activity (p = 0.0003), 9.4 % higher antithrombin activity (p = 0.0004) and 13.5 % lower platelet count (p = 0.02). Other factors and thrombin generation did not differ between the two groups. The ICH survivors were characterized by impaired fibrin polymerization reflected by 10.1 % longer lag phase of the turbidimetry curve (p = 0.0002), decreased fiber density indicated by 11.8 % lower maximum absorbance (p = 0.004), as well as 11.1 % shorter clot lysis time (p = 0.014) and 10.0 % faster increase of maximal D-Dimer levels (p = 0.000001).
CONCLUSIONS: We demonstrated a prohemorrhagic fibrin clot phenotype, along with lower FII, FVII and higher antithrombin activity in adults below 50 years of age who suffered from ICH of unknown cause, which might indicate novel mechanisms contributing to ICH in younger individuals.

BACKGROUND: The SERPINE1 c.-820G (4_5), MTHFR gene variants, and unfavourably altered fibrin clot features, have been suspected to be associated with embolic stroke of undetermined source (ESUS). We investigated the SERPINE1 c.-820G (4_5) gene variants alone and coexisting with MTHFR c.665C > T and c.1286A > C gene variants in relation to thrombophilic factors and plasma fibrin clot properties in Polish patients with ESUS.
METHODS: Unrelated consecutive patients with ESUS (n = 206) were genotyped by TaqMan assay. Thrombophilia screening was performed four weeks or more after a thrombotic event while off oral anticoagulation. Factor VIII (FVIII) activity was determined by a coagulometric assay, while lipoprotein(a) was determined using immunoturbidimetry. We determined fibrin clot permeability (Ks) and clot lysis time (CLT). Apparently healthy individuals without a family history of stroke or venous thromboembolism (n = 30), and patients with a history of atrial fibrillation (n = 25) or carotid artery disease-related stroke (n = 21), served as controls.
RESULTS: Among ESUS patients, the SERPINE1 c.-820G (4_5) minor allele frequency was 0.57. There were no differences in common factors associated with thrombophilia among ESUS patients regarding SERPINE1 variants. The overall prevalence of FVIII > 150IU/dL was 26% (n = 53) and elevated FVIII predominated in SERPINE1 variants carriers (n = 45; 84.9%), including 36 (68%) carriers of MTHFR variant. Moreover, 4.3-fold higher Lp(a) levels along with 50% reduced Ks and 46% prolonged CLT were found in patients with mutant SERPINE1 combined with mutant homozygotes in the MTHFR c.665C > T variant compared to the wild type SERPINE1 combined with mutant homozygotes in the MTHFR c.665C >T (P < 0.001).
CONCLUSIONS: The SERPINE1 c.-820G (4_5) variants carriers have increased FVIII levels, while the SERPINE1 c.-820G (4_5) mutant homozygotes coexisting with MTHFR c.665C > T have more prothrombotic fibrin clot features and elevated Lp(a). Our study underlines the cumulative effect of genetic risk factors in patients with ESUS that might require specific antithrombotic therapy.

Objectives-Metallic elements and fibrin clot properties have been linked to stroke. We examined metallic and nonmetallic elements, fibrin clot lysis time (CLT), and maximum absorbance (Absmax) in relation to ischemic stroke. Design-A case-control study of ischemic stroke patients vs. healthy individuals. Subjects and Methods-Plasma and serum were collected from 260 ischemic stroke patients (45.0% women; age, 68 ± 12 years) and 291 healthy controls (59.7% women; age, 50 ± 17 years). Fibrin CLT and Absmax were measured using a validated turbidimetric assay. Serum elements were quantified by inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES). Data were analyzed by bivariate correlations and multiple or logistic regression. Results-In female stroke patients, copper, lithium, and aluminum were significantly lower compared with controls; in male stroke patients, potassium was lower, and beryllium was elevated. In female and male stroke patients, iron, zinc, nickel, calcium, magnesium, sodium, and silicon were significantly lower, while strontium was elevated. Positive correlations between fibrin clot properties and metals, observed in healthy controls, were lost in ischemic stroke patients. In multivariate regression analysis, fibrin CLT and/or Absmax was associated with zinc, calcium, potassium, beryllium, and silicon in stroke patients and with sodium, potassium, beryllium, and aluminum in controls. In logistic regression analysis, stroke was independently associated with lithium, nickel, beryllium, strontium, boron, and silicon and with sodium, potassium, calcium, and aluminum but not with fibrin CLT/Absmax. Conclusions-Various elements were associated with fibrin clot properties and the risk of ischemic stroke. Lithium, sodium, calcium, and aluminum abrogated the association of fibrin clot properties with ischemic stroke.

Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.

Aminothiols, including cysteine (Cys) and glutathione (GSH) in relation to fibrin clot phenotype were not investigated in patients with venous thromboembolism (VTE) and 5,10-methylenetetrahydrofolate reductase (MTHFR) gene variants. We aimed to explore the associations between MTHFR variants and plasma oxidative stress indicators including aminothiols as well as fibrin clot properties with plasma oxidative status and fibrin clot properties in this group of patients.
In 387 VTE patients the MTHFR c.665C > T and c.1286A > C variants were genotyped, together with chromatographic separation of plasma thiols. We also determined nitrotyrosine levels and fibrin clot properties, including clot permeability (Ks), lysis time (CLT), and fibrin fibers thickness.
There were 193 patients with MTHFR c.665C > T (49.9%) and 214 (55.3%) with c.1286A > C variants. Both allele carriers with total homocysteine (tHcy) levels >15 μM (n = 71, 18.3%), compared to patients with tHcy ≤15 μM had 11.5% and 12.5% higher Cys levels, 20.6% and 34.3% higher GSH levels as well as 28.1% and 57.4% increased nitrotyrosine levels, respectively (all P < 0.05). The MTHFR c.665C > T carriers with tHcy levels >15 μM compared to tHcy ≤15 μM had 39.4% reduced Ks and 9% reduced fibrin fibers thickness (both P < 0.05) with no differences in CLT. In the MTHFR c.1286A > C carriers with tHcy levels >15 μM, Ks was decreased by 44.5%, CLT prolonged by 46.1%, and fibrin fibers thickness was reduced by 14.5% compared to patients with tHcy ≤15 μM (all P < 0.05). Nitrotyrosine levels in MTHFR variants carriers correlated with Ks (r = -0.38, P < 0.05) and fibrin fibers diameter (r = -0.50, P < 0.05).
Our study indicates that patients with MTHFR variants and tHcy >15 μM are characterized by elevated Cys and nitrotyrosine levels associated with prothrombotic fibrin clot properties.

BACKGROUND: Coronary artery disease is associated with impaired clot structure. The aim of this study was to investigate acute phase myocardial infarction (AMI) and provide detailed quantitative analysis of clot ultrastructure.
METHODS: Clot formation and breakdown, pore size, fiber density, fiber radius and protofibril packing were investigated in plasma clots from AMI patients. These data were compared to those from healthy controls.
RESULTS: Analysis on clot formation using turbidity showed increased lag time, suggesting changes in protofibril packing and increased fiber size for AMI patients compared to healthy controls. Additionally, increased average rate of clotting and decreased time to maximum absorbance in AMI patients suggest that clots formed more quickly. Moreover, we observed increased time from max OD to max rate of lysis. Increased fibrinogen and decreased plasminogen in AMI patients were accounted for in represented significant differences. AMI samples showed increased time to 25% and 50% lysis, but no change in 75% lysis, representative of delayed lysis onset, but expediated lysis once initiated. These data suggest that AMI patients formed less porous clots made from more densely packed fibers with decreased numbers of protofibrils, which was confirmed using decreased permeation and increased fiber density, and decreased turbidimetry.
CONCLUSIONS: AMI plasma formed clots that were denser, less permeable, and lysed more slowly than healthy controls. These findings were confirmed by detailed analysis of clot ultrastructure, fiber size, and protofibril packing. Dense clot structures that are resistant to lysis may contribute to a prothrombotic milieu in AMI.

Fibrin clot structure differs between healthy individuals and those following thromboembolic events. Dense and poorly lysable fibrin clots have also been reported in peripheral artery disease. We studied fibrin clot properties and its determinants in individuals with a history of acute lower limb ischemia (ALI) of unknown cause.
In this case-control study, we enrolled 43 patients who experienced ALI of unknown cause, and two age-and sex-matched reference groups: (1) patients with cryptogenic non-lacunar stroke (n = 43) and (2) individuals without any history of thromboembolism (n = 43, control group). Plasma fibrin clot properties, along with thrombin generation and fibrinolysis markers were assessed following ≥3 months of anticoagulation.
Compared with the control group, the ALI group exhibited more compact plasma fibrin clots (13.4% lower permeability [Ks], p = .001), decreased formed clot lysis (12.5% lower D-Drate, p = .001) and unaltered clot lysis potential, along with enhanced thrombin generation potential (49% higher peak thrombin concentration, p < .0001). There were no differences in these variables between ALI and stroke patients. Patients with ALI had slightly higher α2-antiplasmin and lower plasminogen activator inhibitor 1 levels compared with the stroke and control groups (all p < .01).
Patients who experienced ALI of unknown cause display a prothrombotic fibrin clot phenotype, including increased clot density and hypofibrinolysis associated with higher thrombin generation, which might suggest potential benefits from prolonged anticoagulation in this disease.