Measures of endothelial dysfunction in plasma of patients with posttraumatic stress disorder

Posttraumatic stress disorder (PTSD) confers an increased cardiovascular risk. In 14 otherwise healthy patients with PTSD and in 14 age- and gender-matched non-PTSD controls, we investigated whether the categorical diagnosis of PTSD and severity of PTSD symptom clusters (i.e. re-experiencing, avoidance, arousal, and overall score) would be associated with plasma concentrations of three markers of endothelial dysfunction [soluble tissue factor (sTF), von Willebrand factor (VWF), and soluble intercellular adhesion molecule (sICAM)-1]. Compared with controls, patients had significantly higher sTF; this difference became nonsignificant when controlling for psychological distress. VWF and sICAM-1 levels were not significantly different between patients and controls. In the entire sample virtually all PTSD symptom clusters correlated significantly and positively with sTF and VWF but not with sICAM-1. The correlation between symptoms of re-experiencing and sTF was significantly different between patients and controls. Controlling for symptoms of anxiety and depression (i.e. psychological distress) rendered most associations between PTSD symptom clusters and sTF nonsignificant, whereas controlling for age retained significance of associations with VWF. Posttraumatic stress showed a continuous relationship with sTF and VWF, with the former relationship being partly affected by psychological distress. This suggests one mechanism by which posttraumatic stress could contribute to atherosclerosis.

Relation of morning serum cortisol to prothrombotic activity in women with stable coronary artery disease

BACKGROUND: Increased circulating cortisol levels have been associated with severity of atherosclerosis. Low-grade systemic thrombogenicity plays a major role in the initiation and progression of coronary disease. We hypothesized a direct relationship between cortisol and hemostasis factors related to a prothrombotic state in coronary artery disease. METHODS: We measured morning serum cortisol and activated clotting factor VII, fibrinogen, von Willebrand factor antigen, and plasminogen activator inhibitor-1 activity in 285 women (56 +/- 7 years) between 3 and 6 months after an acute coronary event. To test whether the relationship between cortisol and hemostasis factors would be independent, statistical adjustment was made for demographic, biomedical, life style, and psychosocial variables. RESULTS: Higher serum cortisol levels predicted higher fibrinogen (beta = .17, P = .001) and higher von Willebrand factor (beta = .16, P = .008), all independently of covariates, including C-reactive protein, which was also an independent predictor of fibrinogen (beta = .20, P = .001) and von Willebrand factor (beta = .16, P = .004). Higher levels of vital exhaustion were associated with higher levels of activated clotting factor VII independently of covariates and depression (beta = .18, P = .045). Cortisol showed crude correlations with vital exhaustion (r = .14, P = .022) and with depression (r = .13, P = .043) but did not mediate the relationship between psychosocial variables and hemostatic factors. CONCLUSIONS: Morning serum cortisol showed a modest but independent association with prothrombotic activity in women with coronary artery disease suggesting that increased cortisol levels might contribute to atherosclerosis via eliciting a hypercoagulable state.

Autonomic function and prothrombotic activity in women after an acute coronary event

BACKGROUND: The link between decreased heart rate variability (HRV) and atherosclerosis progression is elusive. We hypothesized that reduced HRV relates to increased levels of prothrombotic factors previously shown to predict coronary risk. METHODS: We studied 257 women (aged 56 +/- 7 years) between 3 and 6 months after an acute coronary event and obtained very low frequency (VLF), low frequency (LF), and high frequency (HF) power, and LF/HF ratio from 24-hour ambulatory ECG recordings. Plasma levels of activated clotting factor VII (FVIIa), fibrinogen, von Willebrand factor antigen (VWF:Ag), and plasminogen activator inhibitor-1 (PAI-1) activity were determined, and their levels were aggregated into a standardized composite index of prothrombotic activity. RESULTS: In bivariate analyses, all HRV indices were inversely correlated with the prothrombotic index explaining between 6% and 14% of the variance (p < 0.001). After controlling for sociodemographic factors, index event, menopausal status, cardiac medication, lifestyle factors, self-rated health, metabolic variables, and heart rate, VLF power, LF power, and HF power explained 2%, 5%, and 3%, respectively, of the variance in the prothrombotic index (p < 0.012). There were also independent relationships between VLF power and PAI-1 activity, between LF power and fibrinogen, VWF:Ag, and PAI-1 activity, between HF power and FVIIa and fibrinogen, and between the LF/HF power ratio and PAI-1 activity, explaining between 2% and 3% of the respective variances (p < 0.05). CONCLUSIONS: Decreased HRV was associated with prothrombotic changes partially independent of covariates. Alteration in autonomic function might contribute to prothrombotic activity in women with coronary artery disease (CAD).

Predictors of inflammation in response to anthracycline-based chemotherapy for breast cancer

Although chemotherapy for breast cancer can increase inflammation, few studies have examined predictors of this phenomenon. This study examined potential contributions of demographics, disease characteristics, and treatment regimens to markers of inflammation in response to chemotherapy for breast cancer. Thirty-five women with stage I-III-A breast cancer (mean age 50 years) were studied prior to cycle 1 and prior to cycle 4 of anthracycline-based chemotherapy. Circulating levels of inflammatory markers with high relevance to breast cancer were examined, including C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-alpha), Interleukin-1 receptor antagonist (IL1-RA), vascular endothelial growth factor (VEGF), soluble intercellular adhesion molecule-1 (sICAM-1), Interleukin- (IL-6), soluble P-selectin (sP-selectin), and von Willebrand factor (vWf). Chemotherapy was associated with elevations in VEGF (p < or = 0.01), sICAM-1 (p < or = 0.01), sP-selectin (p < or = 0.02) and vWf (p < or = 0.05). Multiple regression analysis controlling for age and body mass index (BMI) showed that higher post-chemotherapy levels of inflammation were consistently related to higher pre-chemotherapy levels of inflammation (ps < or =0.05) as well as to certain disease characteristics. Post-chemotherapy IL-6 levels were higher in patients who had larger tumors (p < or = 0.05) while post-chemotherapy VEGF levels were higher in patients who had smaller tumors (p < or = 0.05). Post-chemotherapy sP-selectin levels were highest in women who had received epirubicin, cytoxan, 5-fluorouracil chemotherapy (p < or = 0.01). These findings indicate that chemotherapy treatment can be associated with elevations in certain markers of inflammation, particularly markers of endothelial and platelet activation. Inflammation in response to chemotherapy is most significantly related to inflammation that existed prior to chemotherapy but also potentially to treatment regimen and to certain disease characteristics.

Vacuum-assisted closure therapy increases local interleukin-8 and vascular endothelial growth factor levels in traumatic wounds

BACKGROUND: Clinical observations are suggesting accelerated granulation tissue formation in traumatic wounds treated with vacuum-assisted closure (VAC). Aim of this study was to determine the impact of VAC therapy versus alternative Epigard application on local inflammation and neovascularization in traumatic soft tissue wounds. METHODS: Thirty-two patients with traumatic wounds requiring temporary coverage (VAC n = 16; Epigard n = 16) were included. At each change of dressing, samples of wound fluid and serum were collected (n = 80). The cytokines interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), and fibroblast growth factor-2 were measured by ELISA. Wound biopsies were examined histologically for inflammatory cells and degree of neovascularization present. RESULTS: All cytokines were found to be elevated in wound fluids during both VAC and Epigard treatment, whereas serum concentrations were negligible or not detectable. In wound fluids, significantly higher IL-8 (p < 0.001) and VEGF (p < 0.05) levels were detected during VAC therapy. Furthermore, histologic examination revealed increased neovascularization (p < 0.05) illustrated by CD31 and von Willebrand factor immunohistochemistry in wound biopsies of VAC treatment. In addition, there was an accumulation of neutrophils as well as an augmented expression of VEGF (p < 0.005) in VAC wound biopsies. CONCLUSION: This study suggests that VAC therapy of traumatic wounds leads to increased local IL-8 and VEGF concentrations, which may trigger accumulation of neutrophils and angiogenesis and thus, accelerate neovascularization.

VH1-69 germline encoded antibodies directed towards ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

BACKGROUND: Autoantibodies directed towards ADAMTS13 are present in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). Analysis of a set of antibodies derived from two patients with acquired TTP revealed frequent use of the VH1-69 heavy chain gene segment for the assembly of anti-ADAMTS13 antibodies. OBJECTIVE: We explored the ability of two VH1-69 germline gene-encoded antibodies to inhibit the von Willebrand factor (VWF)-processing activity of ADAMTS13 under different experimental conditions. Furthermore, the presence of VH1-69 encoded anti-ADAMTS13 antibodies in 40 patients with acquired TTP was monitored using monoclonal antibody G8, which specifically reacts with an idiotype expressed on VH1-69 encoded antibodies. METHODS AND RESULTS: Binding of the two VH1-69 encoded monoclonal antibodies was dependent on the presence of the spacer domain. Both antibodies inhibited ADAMTS13 activity under static conditions, as measured by cleavage of FRETS-VWF73 substrate and cleavage of VWF multimers. The recombinant antibodies were also capable of inhibiting the processing of UL-VWF strings on the surface of endothelial cells. G8-reactive antibodies directed towards ADAMTS13 were present in plasma of all patients containing anti ADAMTS13 antibodies. CONCLUSIONS: These results suggest that VH1-69 derived antibodies directed towards ADAMTS13 develop in the majority of patients with acquired TTP.

Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood

BACKGROUND: Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting. METHODS: Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads. RESULTS: Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS. CONCLUSIONS: This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ''endothelial protective'' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.

The effect of natural habituation on coagulation responses to acute mental stress and recovery in men

Blood coagulation activation might be one mechanism linking acute mental stress with coronary events. We investigated the natural habituation of coagulation responses and recovery to short-term mental stress. Three times with one-week intervals, 24 men (mean age 47 +/- 7 years) underwent the same 13-min stressor (preparation, job interview, mental arithmetic). During each visit venous blood was obtained four times (baseline, immediately post-stress, 45 min of recovery, 105 min of recovery). Eight blood coagulation parameters were measured at weeks one and three. Acute stress provoked increases in von Willebrand factor antigen, fibrinogen, clotting factor FVII activity (FVII:C), FVIII:C, FXII:C (p's < or = 0.019), and D-dimer (N.S.). All coagulation parameters experienced full recovery except FVIII:C (p = 0.022). Stress did not significantly affect activated partial thromboplastin time and prothrombin time. At all time points FVIII:C and FXII:C levels were significantly higher at week one compared to week three (p's < or = 0.041). Before catheter insertion, systolic blood pressure (p = 0.001) and heart rate (p = 0.026) were relatively higher at week one. Unlike the magnitude of systolic blood pressure response to stress (p = 0.007) and of cortisol recovery from stress (p = 0.002), the magnitude of all coagulation responses to stress and the recovery from stress were similar in week one and week three. Sympathetic activation with anticipatory stress best explained increased baseline activity in FVIII and FXII at week one. An incapacity of the coagulation system to adapt to stress repeats is perhaps a consequence of evolution, but might also contribute to increased coronary risk in some individuals, particularly in those with cardiovascular diseases.

Prothrombotic changes with acute psychological stress: combined effect of hemoconcentration and genuine coagulation activation

INTRODUCTION: Acute psychosocial stress accelerates blood coagulation and elicits hemoconcentration which mechanisms are implicated in acute coronary thrombotic events. We investigated the extent to which the change in prothrombotic measures with acute stress reflects hemoconcentration and genuine activation of coagulation. MATERIAL AND METHODS: Twenty-one middle-aged healthy men underwent three sessions of a combined speech and mental arithmetic task with one-week intervals. Coagulation and plasma volume were assessed at baseline, immediately post-stress, and 45 min post-stress at sessions one and three. Measures of both visits were aggregated to enhance robustness of individual biological stress responses. Changes in eight coagulation measures with and without adjustment for simultaneous plasma volume shift were compared. RESULTS: From baseline to immediately post-stress, unadjusted levels of fibrinogen (p=0.028), clotting factor VII activity (FVII:C) (p=0.001), FVIII:C (p<0.001), FXII:C (p<0.001), and von Willebrand factor (VWF) (p=0.008) all increased. Taking into account hemoconcentration, fibrinogen (p=0.020) and FVII:C levels (p=0.001) decreased, activated partial prothrombin time (APPT) shortened (p<0.001) and prothrombin time (PT) was prolonged (p<0.001). Between baseline and 45 min post-stress, unadjusted (p=0.050) and adjusted (p=0.001) FVIII:C levels increased, adjusted APTT was prolonged (p=0.017), and adjusted PT was shortened (p=0.033). D-dimer levels did not significantly change over time. CONCLUSIONS: Adjustment for stress-hemoconcentration altered the course of unadjusted levels of several prothrombotic factors. After adjustment for hemoconcentration, APPT was shortened immediately post-stress, whereas 45 min post-stress, FVIII:C was increased and PT was shortened. Procoagulant changes to acute stress may reflect both hemoconcentration and genuine activation of coagulation molecules and pathways.

3D scaffolds co-seeded with human endothelial progenitor and mesenchymal stem cells: evidence of prevascularisation within 7 days

Blood supply is a critical issue in most tissue engineering approaches for large defect healing. As vessel ingrowth from surrounding tissues is proven to be insufficient, current strategies are focusing on the neo-vascularisation process. In the present study, we developed an in vitro pre-vascularised construct using 3D polyurethane (PU) scaffolds, based on the association of human Endothelial Progenitor Cells (EPC, CD34+ and CD133+) with human Mesenchymal Stem Cells (MSC). We showed the formation of luminal tubular structures in the co-seeded scaffolds as early as day 7 in culture. These tubular structures were proven positive for endothelial markers von Willebrand Factor and PECAM-1. Of special significance in our constructs is the presence of CD146-positive cells, as a part of the neovasculature scaffolding. These cells, coming from the mesenchymal stem cells population (MSC or EPC-depleted MSC), also expressed other markers of pericyte cells (NG2 and αSMA) that are known to play a pivotal function in the stabilisation of newly formed pre-vascular networks. In parallel, in co-cultures, osteogenic differentiation of MSCs occurred earlier when compared to MSCs monocultures, suggesting the close cooperation between the two cell populations. The presence of angiogenic factors (from autologous platelet lysates) in association with osteogenic factors seems to be crucial for both cell populations' cooperation. These results are promising for future clinical applications, as all components (cells, growth factors) can be prepared in an autologous way.

Congenital thrombotic thrombocytopenic purpura caused by new compound heterozygous mutations of the ADAMTS13 gene

Upshaw-Schulman syndrome (USS) is due to severe congenital deficiency of von Willebrand factor (VWF)-cleaving protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 domains, nr 13) activity resulting in the presence of unusually large forms of VWF in the circulation, causing intravascular platelet clumping and thrombotic microangiopathy. Our patient, a 26-year-old man, had attacks of thrombotic thrombocytopenic purpura (TTP) with thrombocytopenia and a urine dipstick positive for hemoglobin (4+), often as the only sign of hemolytic activity. He had ADAMTS13 activity of <1% of normal plasma without the presence of inhibitors of ADAMTS13. ADAMTS13 deficiency was caused by two new mutations of the ADAMTS13 gene: a deletion of a single nucleotide in exon17 (c. 2042 delA) leading to a frameshift (K681C fs X16), and a missense mutation in exon 25 (c.3368G>A) leading to p.R1123H. This case report confirms the importance of the analysis of the ADAMTS13 activity and its inhibitor in patients who have episodes of TTP, with a very low platelet count and sometimes without the classic biochemical signs of hemolysis.

Hereditary thrombotic thrombocytopenic purpura and the hereditary TTP registry

Hereditary thrombotic thrombocytopenic purpura, Upshaw-Schulman syndrome, ADAMTS13 Hereditary thrombotic thrombocytopenic purpura (TTP), also known as Upshaw-Schulman syndrome, is a rare recessively inherited disease. Underlying is a severe constitutional deficiency of the von Willebrand factor-cleaving protease, ADAMTS13, due to compound heterozygous or homozygous mutations in the ADAMTS13 gene. The clinical picture is variable and more and more patients with an adult-onset are diagnosed. In the majority of countries the only available treatment is plasma, which when administered regularly can efficiently prevent acute disease bouts. The decision to initiate regular prophylaxis is often not easy, as evidence based guidelines and long term outcome data are lacking. Through the hereditary TTP registry (www.ttpregistry.net, ClinicalTrials.gov identifier: NCT01257269), which was initiated in 2006 and is open to all patients diagnosed with Upshaw-Schulman syndrome and their family members, we aim to gain further information and insights into this rare disease, which eventually will help to improve clinical management of affected patients.

Acquired thrombotic thrombocytopenic purpura. Development of an autoimmune response

The von Willebrand factor (VWF)-cleaving metalloprotease, ADAMTS13 (adisintegrin and metalloprotease with thrombospondin type 1 motifs-13) is the only known target of the dysregulated immune response in acquired TTP. Autoantibodies to ADAMTS13 either neutralize its activity or accelerate its clearance, thereby causing a severe deficiency of ADAMTS13 in plasma. As a consequence, size regulation of VWF is impaired and the persistence of ultra-large VWF (ULVWF) multimers facilitates microvascular platelet aggregation causing microangiopathic haemolytic anaemia and ischaemic organ damage. Autoimmune TTP although a rare disease with an annual incidence of 1.72 cases has a mortality rate of 20% even with adequate therapy. We describe the mechanisms involved in ADAMTS13 autoimmunity with a focus on the role of B- and T-cells in the pathogenesis of this disorder. We discuss the potential translation of recent experimental findings into future therapeutic concepts for the treatment of acquired TTP.

Procoagulant reactivity to laboratory acute mental stress in Africans and Caucasians, and its relation to depressive symptoms: the SABPA study.

The risk of cardiovascular disease is dramatically increasing in Africans (black). The prothrombotic stress response contributes to atherothrombotic disease and is modulated by depressive symptoms. We examined coagulation reactivity to acute mental stress and its relation to psychological well-being in Africans relative to Caucasians (white). A total of 102 African and 165 Caucasian school teachers underwent the Stroop Color-Word Conflict test. Circulating levels of von Willebrand factor (VWF) antigen, fibrinogen, and D-dimer were measured before and after the Stroop. Cardiovascular reactivity measures were also obtained. All participants completed the Patient Health Questionnaire-9 and the General Health Questionnaire-28 for the assessment of depressive symptoms and total psychological distress, respectively. After controlling for covariates, resting levels of VWF, fibrinogen, and D-dimer were higher in Africans than in Caucasians (all p-values ≤0.006). Depressive symptoms and psychological distress were not significantly associated with resting coagulation measures. Stress reactivity in VWF (p<0.001) and fibrinogen (p=0.016), but not in D-dimer (p=0.27), were decreased in Africans relative to Caucasians with Africans showing greater reactivity of total peripheral resistance (p=0.017). Depressive symptoms, but not general psychological distress, were associated with greater VWF increase (p=0.029) and greater fibrinogen decrease (p=0.030) in Africans relative to Caucasians. In conclusion, Africans showed greater hypercoagulability at rest but diminished procoagulant reactivity to acute mental stress when compared with Caucasians. Ethnic differences in the vascular adrenergic stress response might partially explain this finding. Depressive symptoms were associated with exaggerated VWF reactivity in Africans relative to Caucasians. The clinical implications of these findings for Africans need further study.

The effect of desmopressin on platelet function: a selective enhancement of procoagulant COAT platelets in patients with primary platelet function defects.

1-deamino-8-d-arginine vasopressin (desmopressin [DDAVP]) is clinically efficacious in patients with mild platelet function disorders but it is not known which mechanisms mediate this effect. Our aim was to evaluate the impact of in vivo DDAVP administration in these patients. We assessed von Willebrand factor (VWF), factor VIII, platelet activation and aggregation, platelet-dependent thrombin generation, and platelet intracellular Na(+)/Ca(2+) fluxes, before and 2 and 4 hours after DDAVP (0.3 µg/kg). We found (1) no significant changes for P-selectin expression, PAC-1 binding, δ-granule content and secretion, and platelet-aggregation; (2) significant decreases of secretion of α-granules and GPIIb-IIIa activation induced by adenosine 5'-diphosphate, convulxin, and thrombin; (3) significant increases of procoagulant platelets induced by convulxin/thrombin and platelet-dependent thrombin generation; and (4) significant increases of intracellular Na(+)/Ca(2+) concentrations. We show that in vivo DDAVP selectively and markedly enhances the ability to form procoagulant platelets and increases platelet-dependent thrombin generation by enhancing Na(+)/Ca(2+) mobilization. This report indicates that the beneficial hemostatic effect of DDAVP is not limited to an increase in large VWF multimers. An enhancement of platelet procoagulant activity appears to be an additional and (at least in platelet disorders) -possibly clinically relevant mechanism of DDAVP's action.