Diego Mezzano

Inflammation, not hyperhomocysteinemia, is related to oxidative stress and hemostatic and endothelial dysfunction in uremia

BACKGROUND Several cardiovascular risk factors are present in patients with chronic renal failure (CRF), among which are systemic inflammation and hyperhomocysteinemia. Increased oxidative stress, endothelial activation/dysfunction, and coagulation activation are considered integral components of the inflammatory response, but have also been proposed as mediators of plasma homocysteine (tHcy)-induced cell damage. Using correlation analysis, we assessed the relative contributions of inflammation and hyperhomocysteinemia in the abnormal oxidative stress, endothelial activation/dysfunction, and hemostasis activation in patients with CRF. METHODS The relationships of inflammatory proteins and tHcy with plasma markers of these processes were studied in 64 patients with CRF (serum creatinine 526 +/- 319 micromol/L) on conservative treatment, comparing the results with healthy controls (N = 15 to 40, depending on the measured variable) of similar sex and age. RESULTS Patients had significant increases in inflammatory cytokines (TNF-alpha and IL-8) and acute-phase proteins (C-reactive protein, fibrinogen and alpha1-antitrypsin). tHcy was increased in 87.5% of patients (mean = 27.1 micromol/L, range 6.5 to 118). Patients had significant increases in (1) indices of oxidative stress: TBARS (thiobarbituric acid-reactive species), a marker of lipid peroxidation and AOPP (advanced oxidation protein products), a marker of protein oxidation; (2) endothelial cell markers such as von Willebrand factor (vWF:Ag), soluble ICAM-1 and soluble thrombomodulin (sTM); (3) markers of intravascular thrombin generation: thrombin-antithrombin complexes (TAT) and prothrombin fragment F(1+2) (PF(1+2)); and (4) indices of activation of fibrinolysis: plasmin-antiplasmin complexes (PAP), fibrin degradation products (FnDP) and fibrinogen degradation products (FgDP). tHcy was significantly correlated with plasma creatinine (r = 0.29, P < 0.018) and with serum folate (r = -0.38, P < 0.002). However, no significant correlations were observed between tHcy and TBARS, AOPP, vWF:Ag, sICAM-1, sTM, TAT, F(1+2), sTF, PAP, FnDP, and FgDP. Conversely, acute-phase proteins showed significant, positive correlations with most markers of oxidative stress, endothelial dysfunction and hemostatic activation. CONCLUSIONS Systemic inflammation, which is closely associated with augmented oxidative stress, endothelial cell dysfunction and hemostatic activation, emerges as a major cardiovascular risk factor in CRF. tHcy is unrelated to these events. Thus, alternative mechanisms through which hyperhomocysteinemia could predispose to vascular lesion and thrombotic events in CRF needs to be investigated.

Template bleeding time and PFA-100 have low sensitivity to screen patients with hereditary mucocutaneous hemorrhages: comparative study in 148 patients.

Summary.  Objectives and patients: We compared the template bleeding time (BT) and closure time (CT) in the PFA‐100® as screening tests in 148 consecutive patients with unequivocal mucocutaneous bleeding and positive family history. Exclusion criteria: drug intake, concomitant diseases including minor infections, low platelet count, diseases of secondary hemostasis.Results: Type 1 von Willebrand disease (VWD‐1) was diagnosed in 26 patients, primary platelet secretion defect (PSD) in 33, VWD‐1 + PSD in nine, whereas 80 patients did not comply with the criteria for known hemostatic disorders (UD, unknown diagnosis). BT and CT were prolonged in 35.8% and 29.7% of all the patients, respectively (P = 0.23). Sensitivity increased to 48% if an abnormality of BT and/or CT was considered. Same comparisons for BT and CT in each diagnostic category were, respectively: 42 vs. 61.5% in VWD‐1 (P = 0.18), 42 vs. 24% in platelet secretion defects (P = 0.11), 67 vs. 89% in VWD‐1 + PSD (P = 0.50), and 27.5 vs. 15% in UD (P = 0.06). Conclusion: Both tests were relatively insensitive and not significantly different in detecting incoming patients with mucocutaneous hemorrhages. In patients with VWD‐1, the PFA‐100® performed slightly better, whereas the opposite occurred in those patients with platelet secretion defects. In the UD group, both tests lost sensitivity, but the BT detected 1.8 times more patients than the PFA‐100®. Given the large proportion of undiagnosed bleeders and the overall low sensitivity of these tests, clinical decisions still rely on the medical history and etiological diagnosis of the bleeding disorder.

Endothelial cell markers in chronic uremia: relationship with hemostatic defects and severity of renal failure.

Plasma von Willebrand factor antigen, soluble thrombomodulin, and tissue factor were increased in 31 patients with severe chronic renal failure (creatinine clearance <20 ml/min) under conservative treatment, whereas plasminogen activator inhibitor antigen did not differ significantly from healthy controls. No correlation among plasma levels of these proteins was found. Three patterns of relationship between endothelial cell markers and hemostatic defects were identified: 1) Plasma thrombomodulin, a marker of endothelium damage, was found an independent predictor of bleeding time and platelet aggregation, and secretion defects, and was also related to the severity of renal failure; 2) von Willebrand factor antigen, an index of endothelial cell activation and secretion, was significantly correlated with intravascular markers of thrombin and plasmin generation and with platelet adenosine triphosphate content, but not with plasma creatinine levels; and 3) tissue factor and plasminogen activator inhibitor antigen levels were not statistically correlated with the diverse hemostatic defects. Activation of coagulation and fibrinolysis, secondary to endothelial cell activation, appearing early during the evolution of chronic renal failure, is pathogenically related to the platelet dysfunction, and probably to development of atherosclerosis and thrombotic events in this disease. The progression of chronic renal failure, through endothelial cell damage, would lead to aggravation of the platelet functional defect potentiating the hemorrhagic risk.

Cardiovascular Risk Factors in Vegetarians: Normalization of Hyperhomocysteinemia with Vitamin B12 and Reduction of Platelet Aggregation with n-3 Fatty Acids

Hyperhomocysteinemia in association with vitamin B12 deficiency, and increased platelet aggregation, probably due to dietary lack of n-3 fatty acids, constitute cardiovascular risk factors frequently observed in vegetarians. We tested if administration of vitamin B12 normalizes the concentration of total plasma homocysteine, and if intake of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) fatty acids modulates platelet function in a population of lactoovovegetarians. One week after a single intramuscular injection of cyanocobalamin (10000 μg) in 18 individuals, serum vitamin B12 increased from 149±63 pg/mL to 532±204 pg/mL (p<0.0001) and total tHcy dropped from 12.4±4.7 to 7.9±3.1 μmol/L (p<0.0001). Ten of fourteen of these vegetarians completed an 8-week supplementation with 700 mg/day of each eicosapentaenoic and docosahexaenoic acids. Increased incorporation of these fatty acids into plasma lipids was observed in all of them, together with a significant reduction in maximum percentage or slope of platelet aggregation with all the agonists tested (ADP, epinephrin, collagen, arachidonic acid). No significant change in bleeding time was observed after n-3 fatty acid trial. Supplementation with vitamin B12 and n-3 fatty acids corrects hyperhomocysteinemia and reduces platelet reactivity to agonists in vegetarians. Whether this supplementation improves the already reduced cardiovascular morbidity and mortality associated with vegetarian diet has yet to be demonstrated.

Glycoprotein Ib/IX complex is the target in rifampicin-induced immune thrombocytopenia.

Thrombocytopenia is a major adverse effect of several drug treatments. Rifampicin has been recognized as a cause of immune thrombocytopenia during intermittent high‐dose therapy. We characterized the antibody of a patient who presented with purpura and thrombocytopenia during treatment of tuberculosis with rifampicin. Drug‐dependent binding of the antibody to platelets was demonstrated by flow cytometry. In a glycoprotein‐specific immunoassay, the binding epitope of the IgG antibody was found in the glycoprotein Ib/IX complex, using four different monoclonal antibodies (mAbs) against various epitopes on the GPIb/IX complex, as well as mAbs against GPIIb/IIIa, GPIa/IIa and GPIV. By immunoprecipitation of biotin‐labelled platelets, reactivity of the antibody with GPIb/IX was found only in the presence of the drug. These findings clearly demonstrate that rifampicin induces the formation of drug‐dependent antibodies capable of causing thrombocytopenia. The binding site of the rifampicin‐dependent antibody, located in the GPIb/IX complex, seems to be a favoured target for antibodies induced by different drugs.

Increased activation of protein C, but lower plasma levels of free, activated protein C in uraemic patients: relationship with systemic inflammation and haemostatic activation

Chronic renal failure (CRF) courses with both systemic inflammatory reaction and haemostatic activation. We explored the relationship of these processes with plasma levels of free, activated protein C (APC) and complexes of APC with its inhibitors in patients with CRF under conservative treatment. Plasma concentrations of inflammatory cytokines [tumour necrosis factor alpha (TNFα) and interleukin 8], acute‐phase proteins (C‐reactive protein, fibrinogen, α1‐anti‐trypsin and von Willebrand factor), and markers of haemostatic activation (thrombin–anti‐thrombin complexes, plasmin–anti‐plasmin complexes, and fibrin and fibrinogen degradation products) were higher in patients than in controls. Inflammatory and haemostatic markers were significantly and positively correlated. Total plasma APC and APC:α1‐anti‐trypsin (α1AT) complexes were 44% and 75% higher in patients than in controls (P = 0·0001), whereas free APC was 20% lower (P < 0·015). No significant difference was observed in APC:protein C inhibitor (PCI) complexes between both groups. The free/total APC ratio was significantly lower in patients than in controls (P < 0·0001). Total plasma APC and APC:α1AT were positively correlated with activation markers of haemostasis and acute‐phase proteins, whereas free APC was inversely correlated with plasma levels of creatinine, acute‐phase proteins and fibrin degradation products (FnDP). Systemic inflammation and activation of haemostasis are interrelated processes in CRF. APC generation was increased in response to elevated thrombin production, but the inflammatory reaction, associated with increased synthesis of α1AT, reduced its anticoagulant effect. Lower free plasma APC in CRF may be pathogenically associated with atherothrombosis, a major cause of death in this disease.

Distinctive Effects of Red Wine and Diet on Haemostatic Cardiovascular Risk Factors

The aim of this study was to compare the effects of Mediterranean-type diet (MD), high-fat diet (HFD), and red wine supplementation on plasma concentration of emergent haemostatic cardiovascular risk factors (HCVRF) and on variables of primary haemostasis (bleeding time, plasma von Willebrand factor and platelet aggregation/secretion). In a controlled prospective intervention study, two groups (21 healthy males each) received either MD or HFD during 90 days. Between days 30-60, both diets were supplemented with 240 ml/ day of red wine. After adjusting by baseline values, MD was associated with: lower plasma fibrinogen (p =0.03), factor VIIc (p=0.034) and factor VIIIc (p=0.0057); higher levels of protein S (p=0.013); longer bleeding time (p=0.017); and marginal increases in platelet serotonin aggregation and secretion after stimulation with epinephrine. Red wine supplementation, in both diets, resulted in decreased plasma fibrinogen (p=0.001) and factor VIIc (p=0.05), and in increased t-PA (p=0.01) and PAI-1 (p=0.0003). The effects of wine on antithrombin III (p=0.01) were divergent: there was a decrease in the HFD group but it increased slightly in the MD group. No effects of diet or wine were detected in plasma protein C, C-reactive protein or von Willebrand factor. BT did not change significantly with wine supplementation. Wine intake resulted in a significant increase in ex vivo platelet aggregation and secretion after stimulation with collagen (1 and 2 microg/ml, p < or = 0.01). MD and moderate consumption of red wine have complementary, mostly beneficial effects on haemostatic CV risk factors. The longer BT in individuals on MD, obtained independently of red wine, denotes less interaction of platelets with the vascular wall, which could be beneficial from the point of view of CV risk.

Thrombin Generation in Platelet-Poor Plasma Is Normal in Patients with Hereditary Mucocutaneous Haemorrhages

Mild hereditary bleeding disorders presenting with mucocutaneous haemorrhages are usually difficult to diagnose. We measured thrombin generation in platelet-poor plasma (TG-PPP) in 206 patients with a clinically unequivocal bleeding tendency: 45 with von Willebrand disease (vWD), 49 with platelet aggregation/secretion defects (PASD), 10 with a combination of both and 102 who did not fit the diagnostic criteria for any known haemostatic disorder. TG-PPP was not significantly different from controls in all patient groups, indicating that an abnormality in the plasmatic clotting system is unlikely to contribute to the bleeding in patients with type 1 vWD and PASD. In patients with undiagnosed mild hereditary bleeding disorders, there must be other mechanisms which explain the abnormal haemorrhagic tendency, most likely as yet unrecognized defects in platelet-vessel wall interaction. As a next step we plan to investigate thrombin generation in PRP.

Clot lysis time in platelet-rich plasma: method assessment, comparison with assays in platelet-free and platelet-poor plasmas, and response to tranexamic acid.

Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p < 0.0001). PFP- and PPP-, but more significantly PRP-CLT, were positively correlated with age and plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p < 0.001). All these CLT assays had no significant correlations with platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is likely explained by the display of platelet pro-fibrinolytic effects. Focused research is needed to disclose the mechanisms for the relationship between CLT and plasma cholesterol and its potential pathophysiologic and clinical relevance.

Influence of the Thr325Ile polymorphism on procarboxypeptidase U (thrombin‐activable fibrinolysis inhibitor) activity‐based assays

Fig. S1. Flow cytometric analysis of peripheral blood for circulating endothelial cell (CEC) capture. (A, B, C). Typical forward scatter (FSC)/side scatter (SSC) profile of blood leukocytes [in this case, a patient with acute myocardial infarction (AMI)], demonstrating sequential gating strategy for mature CEC enumeration. G2 represents a gate to exclude all non-relevant CD45 + cells, and cells of high SSC, R1 represents CD45 ) /146 + /34 + CECs, and R2 represents CD34 + progenitor cells. (D, E, F) Typical FSC/SSC plots from three different groups of subjects, demonstrating FSC/ SSC characteristics of CECs (in bold for clarity). Note that the majority of CECs are comparable to normal peripheral blood mononuclear cells (PBMCs). (G) A typical scatter-plot of an unstained blood sample (from an AMI patient) spiked with cultured HUVECs (highlighted in bold for clarity), demonstrating their considerably different FSC/SSC characteristics compared to PBMCs (A). (H) In the same unstained sample, unstained HUVECs (boxed area) also possess a greater degree of autofluorescence compared with other blood leukocytes (fluorescence in FL1); compare this with a similarly unstained sample without HUVECs (I, J). (K) A representative plot of a pure unstained HUVEC population, further demonstrating autofluorescence in FL2 and FL3 channels, and (L) when stained specifically for CD146 and CD34.