Circulating microaggregates as biomarkers for the Post-COVID syndrome.

CoVID-19 can develop into Post-COVID syndrome of potentially high morbidity, with procoagulation and reactivation of dormant viral infections being hypothesized pathophysiological mechanisms. We report on a patient suffering from fatigue, post exertional malaise, pain and neurological symptoms as a consequence of the second CoVID infection. Using live confocal microscopy on native whole blood samples we detected microaggregates of thrombocytes, leukocytes and plasma proteins in peripheral blood. In addition, there was specific cellular immunological reactivity to EBV. Upon anticoagulatory and virustatic pharmacological therapy we observed dissolution of microaggregates and significant stable clinical remission. We suggest to consider circulating microaggregates as a morphological indicator of chronic post-COVID syndrome.

A comparison of the new ROTEM® sigma with its predecessor, the ROTEMdelta.

Thromboelastometry point-of-care coagulation testing facilitates optimised management of bleeding. Previous thromboelastometry systems required the blood sample and liquid reagents to be pipetted in several manual steps by trained personnel. The ROTEMsigma coagulation analyser is a fully automated point-of-care device. We aimed to assess the reference ranges of the new device and to compare the results with those of the predecessor device, the ROTEMdelta. We took blood from healthy volunteers and from hyper- or hypocoagulable patients; blood samples from healthy volunteers served to determine reference ranges for the most important parameters for the ROTEMsigma: CTEXTEM 48-61 s; A5EXTEM 30-51 mm; MCFEXTEM 54-70 mm; CTINTEM 138-174 s; MCFINTEM 51-67 mm and MCFFIBTEM 5-24 mm. We then used blood samples from patients to compare the results obtained between the old and the new device. We found a strong correlation between the same tests performed on two ROTEMsigma devices and between the ROTEMsigma and the ROTEMdelta with respect to the determination of thromboelastometry parameters of hyper- and hypocoagulable patients (all p < 0.001 and R > 0.8). Performance evaluation for the ROTEMsigma device showed very high precision (R > 0.99, p < 0.001). Our reference ranges can serve as an important aid for other hospitals using this new device.

Novel antiplatelet role for a protein disulfide isomerase-targeted peptide: evidence of covalent binding to the C-terminal CGHC redox motif.

Essentials Inhibitors of protein disulfide isomerase (PDI) have been considered a new antithrombotic class. CxxC is a PDI-targeted peptide that has been previously shown to inhibit its reductase activity. CxxC binds to surface PDI and inhibits ADP- and thrombin-evoked platelet activation and aggregation. CxxC binds to Cys400 on CGHC redox motif of PDI a' domain, a site for PDI prothrombotic activity. SUMMARY: Background Protein disulfide isomerase (PDI) plays a major role in platelet aggregation, and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on a PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized that this peptide would also inhibit platelet aggregation by association with surface PDI. Methods Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. These peptides were tested under platelet aggregation and flow cytometry protocols to identify their possible antiplatelet activity. We labeled membrane free thiol and electrospray ionization liquid chromatography tandem mass spectrometry to test for an interaction. Results CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. CxxC decreased aIIbb3 activation, but had no effect on the other markers. CxxC also decreased cell surface PDI pulldown without interfering with the total thiol protein content. Finally, we detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 through mass spectrometry. Interestingly, CxxC did not react with oxidized PDI. Discussion CxxC has consistently shown its antiplatelet effects, both in PRP and washed platelets, corroborated by decreased aIIbb3 activation. The probable mechanism of action is through a mixed dissulphide bond with Cys400 of PDI, which has been shown to be essential for PDI's actions. Conclusion In summary, our data support antiplatelet activity for CxxC through binding to Cys400 in the PDI a0 domain, which can be further exploited as a model for sitedriven antithrombotic agent development.

Fibrinogen supplementation ex vivo increasesclot firmness comparable to platelet transfusion in thrombocytopenia.

BACKGROUND: Fibrinogen concentrate can improve clot firmness and offers a better safety profile than platelet concentrates. Reduction or avoidance of blood transfusions represents a strategy to reduce associated risks. We investigated whether supplementation of fibrinogen concentrate ex vivo can compensate for clot strength as compared with platelet transfusion in vivo METHODS: One hundred patients in need of platelet transfusion (PT) were enrolled. Blood samples were collected immediately before PT and at 1 h and 24 h after PT. Fibrinogen concentrate was added to these citrated whole blood samples at concentrations of 50, 100, 200 and 400 mg kg-1 and the maximum clot firmness (MCF) was analysed using ROTEM thromboelastometry. RESULTS: Fibrinogen supplementation increased MCF significantly and dose-dependently before and after PT. The effect of fibrinogen concentrate (equivalent to doses of 100 and 200 mg kg-1) ex vivo was comparable to that of PT in vivo, whereas 400 mg kg-1 fibrinogen significantly improved MCF compared with PT (P < 0.001). CONCLUSIONS: Fibrinogen concentrate can match the effect of PT on MCF in thrombocytopenia. This potential alternative haemostatic intervention should be evaluated in clinical trials.

Ex vivo reversal of effects of rivaroxaban evaluated using thromboelastometry and thrombin generation assay.

BACKGROUND: In major bleeding events, the new direct oral anticoagulants pose a great challenge for physicians. The aim of the study was to test for ex vivo reversal of the direct oral anticoagulant rivaroxaban with various non-specific reversal agents: prothrombin complex concentrate (PCC), activated prothrombin complex concentrate (aPCC), recombinant activated factor VII (rFVIIa), and fibrinogen concentrate (FI). METHODS: Blood was obtained from healthy volunteers and from patients treated with rivaroxaban. Blood samples from healthy volunteers were spiked with rivaroxaban to test the correlation between rivaroxaban concentration and coagulation tests. Patient blood samples were spiked with various concentrations of the above-mentioned agents and analysed using thromboelastometry and thrombin generation. RESULTS: When added in vitro, rivaroxaban was significantly (P<0.05) correlated with ROTEM® thromboelastometry EXTEM (extrinsic coagulation pathway) clotting time (CT), time to maximal velocity (MaxV-t), and with all measured thrombin generation parameters. In vivo, CT, MaxV-t, lag time, and peak thrombin generation (Cmax) were significantly correlated with rivaroxaban concentrations. Regarding reversal of rivaroxaban, all tested agents significantly (P<0.05) reduced EXTEM CT, but to different extents: rFVIIa by 68%, aPCC by 47%, PCC by 17%, and FI by 9%. Only rFVIIa reversed EXTEM CT to baseline values. Both PCC (+102%) and aPCC (+232%) altered overall thrombin generation (area under the curve) and increased Cmax (+461% for PCC, +87.5% for aPCC). CONCLUSIONS: Thromboelastometry and thrombin generation assays do not favour the same reversal agents for rivaroxaban anticoagulation. Controlled clinical trials are urgently needed to establish doses and clinical efficacy of potential reversal agents. CLINICAL TRIAL REGISTRATION: EudracCT trial no. 213-00474-30.

Fibrinogen concentrate improves clot strength in patients with haematological malignancies requiring platelet transfusion.

BACKGROUND: Patients with bone marrow failure secondary to chemotherapy often develop thrombocytopenia and require platelet transfusion. Fibrinogen plays an important role in platelet aggregation and the establishment of the primary haemostatic plug. OBJECTIVES: To compare the effects of in vivo platelet transfusion on clot firmness in thrombocytopenic patients with in vitro-performed fibrinogen concentrate substitution. MATERIALS AND METHODS: Thirty patients with haematological malignancy admitted for platelet transfusion were included. Haemostatic effects from platelet transfusion and ex vivo addition of fibrinogen concentrate at three different doses were evaluated by thromboelastometry, with clot firmness as the primary endpoint (A30 ExTEM assay). Secondary endpoints were other thromboelastometry parameters, thrombin generation parameters, activated partial thromboplastin time (APTT), prothrombin time PT, fibrinogen and factor XIII levels and a clinical bleeding score. RESULTS: Twenty patients (66%) had clinical bleeding signs by prior to transfusion. Platelets increased from 17 (range, 1-109) to 40 (range 2-139) × 10(9) L(-1) following transfusion, with a median corrected count increment of 16·7 (range, 0·8-43·5). The A30 value increased significantly by platelet transfusion from 35 ± 11 to 47 ± 10 mm, with no changes in thrombin generation. Fibrinogen concentrate dose-dependently increased A 30 (to 43 ± 10, 49 ± 9 and 50 ± 9 mm, respectively) and reduced parameters of thrombin generation at high doses. Platelet transfusion, together with fibrinogen concentrate, further increased clot firmness. APTT and PT were within normal range, whereas fibrinogen levels were slightly elevated. CONCLUSION: Fibrinogen concentrate increased clot firmness to the same degree as platelet transfusion in patients with low platelet count requiring platelet transfusion.

Rediscovering the wound hematoma as a site of hemostasis during major arterial hemorrhage.

BACKGROUND: Treatments for major internal bleeding after injury include permissive hypotension to decrease the rate of blood loss, intravenous infusion of plasma or clotting factors to improve clot formation, and rapid surgical hemostasis or arterial embolization to control bleeding vessels. Yet, little is known regarding major internal arterial hemostasis, or how these commonly used treatments might influence hemostasis. OBJECTIVES: (i) To use a swine model of femoral artery bleeding to understand the perivascular hemostatic response to contained arterial hemorrhage. (ii) To directly confirm the association between hemodynamics and bleeding velocity. (iii) To observe the feasibility of delivering an activated clotting factor directly to internal sites of bleeding using a simplified angiographic approach. METHODS: Ultrasound was used to measure bleeding velocity and in vivo clot formation by elastography in a swine model of contained femoral artery bleeding with fluid resuscitation. A swine model of internal pelvic and axillary artery hemorrhage was also used to demonstrate the feasibility of local delivery of an activated clotting factor. RESULTS: In this model, clots formed slowly within the peri-wound hematoma, but eventually contained the bleeding. Central hemodynamics correlated positively with bleeding velocity. Infusion of recombinant human activated factor VII into the injured artery near the site of major internal hemorrhage in the pelvis and axillae was feasible. CONCLUSIONS: We rediscovered that clot formation within the peri-wound hematoma is an integral component of hemostasis and a feasible target for the treatment of major internal bleeding using activated clotting factors delivered using a simplified angiographic approach.

Usefulness of standard plasma coagulation tests in the management of perioperative coagulopathic bleeding: is there any evidence?

Standard laboratory coagulation tests (SLTs) such as prothrombin time/international normalized ratio or partial thromboplastin time are frequently used to assess coagulopathy and to guide haemostatic interventions. However, this has been challenged by numerous reports, including the current European guidelines for perioperative bleeding management, which question the utility and reliability of SLTs in this setting. Furthermore, the arbitrary definition of coagulopathy (i.e. SLTs are prolonged by more than 1.5-fold) has been questioned. The present study aims to review the evidence for the usefulness of SLTs to assess coagulopathy and to guide bleeding management in the perioperative and massive bleeding setting. Medline was searched for investigations using results of SLTs as a means to determine coagulopathy or to guide bleeding management, and the outcomes (i.e. blood loss, transfusion requirements, mortality) were reported. A total of 11 guidelines for management of massive bleeding or perioperative bleeding and 64 studies investigating the usefulness of SLTs in this setting were identified and were included for final data synthesis. Referenced evidence for the usefulness of SLTs was found in only three prospective trials, investigating a total of 108 patients (whereby microvascular bleeding was a rare finding). Furthermore, no data from randomized controlled trials support the use of SLTs. In contrast, numerous investigations have challenged the reliability of SLTs to assess coagulopathy or guide bleeding management. There is actually no sound evidence from well-designed studies that confirm the usefulness of SLTs for diagnosis of coagulopathy or to guide haemostatic therapy.

[Management of postpartum hemorrhage (PPH): algorithm of the interdisciplinary D-A-CH consensus group PPH (Germany - Austria - Switzerland)]. / Management der postpartalen Blutung (PPH): Algorithmus der Interdisziplinären D-A-CH-Konsensusgruppe PPH (Deutschland - Österreich - Schweiz).

Postpartum hemorrhage (PPH) is one of the main causes of maternal deaths even in industrialized countries. It represents an emergency situation which necessitates a rapid decision and in particular an exact diagnosis and root cause analysis in order to initiate the correct therapeutic measures in an interdisciplinary cooperation. In addition to established guidelines, the benefits of standardized therapy algorithms have been demonstrated. A therapy algorithm for the obstetric emergency of postpartum hemorrhage in the German language is not yet available. The establishment of an international (Germany, Austria and Switzerland D-A-CH) "treatment algorithm for postpartum hemorrhage" was an interdisciplinary project based on the guidelines of the corresponding specialist societies (anesthesia and intensive care medicine and obstetrics) in the three countries as well as comparable international algorithms for therapy of PPH.The obstetrics and anesthesiology personnel must possess sufficient expertise for emergency situations despite lower case numbers. The rarity of occurrence for individual patients and the life-threatening situation necessitate a structured approach according to predetermined treatment algorithms. This can then be carried out according to the established algorithm. Furthermore, this algorithm presents the opportunity to train for emergency situations in an interdisciplinary team.

Coagulation Day 2010: an Austrian survey on the routine of thromboprophylaxis in intensive care.

PURPOSE: Venous thromboembolism (VTE) is a common but often overlooked life-threatening complication of critical illness. The aim of this cross-sectional survey was to assess current practice of thromboprophylaxis as well as adherence to international guidelines. METHODS: After ethics committee approval, all intensive care units in Austrian hospitals treating adult patients were invited to participate in this web-based survey. Anonymized data on each patient treated at the participating intensive care units on Coagulation Day 2010 were collected using an electronic case report form. Risk assessment, choice and monitoring of anticoagulants, means of mechanical prophylaxis, and demographic data were recorded. RESULTS: Data from 325 critically ill patients were collected. Patients had a median of four risk factors for thrombosis and 6 % suffered from VTE. Of the 325 patients, 80 % received low molecular weight heparins subcutaneously, 10 % received unfractionated heparin intravenously, 1 % received alternative anticoagulants and 9 % received no pharmacological prophylaxis. Mechanical prophylaxis was used in 49 % with a predominant use of graduated compression stockings. In 39 % a combination of pharmacological and mechanical prophylaxis was applied and 5 % received no prophylaxis at all. Overall guideline adherence was 40 % on Coagulation Day 2010. CONCLUSION: Current practice of thromboprophylaxis is predominantly based on the administration of low molecular weight heparins prescribed at rather arbitrary doses without a discernible relationship to drug monitoring, thromboembolic risk factors, vasopressor use or fluid balance. The use of mechanical prophylaxis, evaluation of risk scores and overall guideline adherence must be further encouraged by education, training and communication.

Emerging treatment strategies for trauma-induced coagulopathy.

BACKGROUND: Trauma-induced coagulopathy has a multifactorial aetiology. Coagulopathy is related to blood loss including consumption of clotting factors and platelets and haemodilution. Additionally hyperfibrinolysis, hypothermia, acidosis and metabolic changes affect the coagulation system. METHODS: This is a review of pathophysiology and new treatment strategies for trauma-induced coagulopathy. RESULTS: Paradigms are actively changing and there is still a shortage of data. The aim of any haemostatic therapy is to control bleeding and minimize blood loss and transfusion requirements. Transfusion of allogeneic blood products as well as trauma-induced coagulopathy cause increased morbidity and mortality. Current opinion is based on present studies and results from small case series, combined with findings from experimental studies in animals, in vitro studies and expert opinions, as opposed to large, randomized, placebo-controlled studies. A summary of new and emerging strategies, including medical infusion and blood products, to beneficially manipulate the coagulation system in the critically injured patient is suggested. CONCLUSION: Future treatment of trauma-induced coagulopathy may be based on systemic antifibrinolytics, local haemostatics and individualized point-of-care-guided rational use of coagulation factor concentrates such as fibrinogen, prothrombin complex concentrate, recombinant factor VIIa and factor XIII. The authors speculate that timely and rational use of coagulation factor concentrates will be more efficacious and safer than ratio-driven use of transfusion packages of allogeneic blood products. Copyright © 2011 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

Unconditioned and conditioned effects of intranasally administered insulin vs placebo in healthy men: a randomised controlled trial.

AIMS/HYPOTHESIS: In humans, the intranasal route allows insulin to reach the brain while maintaining peripheral euglycaemia. Our aims were to examine acute (unconditioned) effects of central insulin on normal-range blood glucose and hormones in men, and to find out whether the effects of intranasal insulin can be learnt via classical conditioning. METHODS: In a randomised controlled trial, 32 healthy normal-weight men (mean age 24.2 [SEM 0.5], mean BMI 22.4 [0.3]) received a conditioned stimulus (CS) and six administrations of either soluble H-insulin 100 (20 U [0.2 ml]; group 1; n = 16) or vehicle (0.2 ml; group 2; n = 16) on day 1. The CS was the tarry smell of meta-cresol (used as a stabilising vehicle in many insulin preparations and placebos). On day 2, all participants received the CS and six administrations of placebo. Participants and experimenters were blinded to group assignment. Sixteen individuals were randomised to and analysed in each group. Participants were sequentially numbered for group allocation. The main outcome measures were blood glucose and insulin, expressed as cumulative difference-from-baseline changes. RESULTS: While maintaining euglycaemia, intranasal insulin induced an increase of peripheral insulin on day 1 (group 1, 17.78 [21.88] pmol/l vs group 2, -10.24 [9.42] pmol/l), and also on day 2 when the CS was given with placebo (group 1, 12.53 [5.57] pmol/l vs group 2, -5.51 [6.16] pmol/l). Moreover, a moderate reduction of blood glucose on day 1 (group 1, -0.54 [0.36] mmol/l vs group 2, 0.58 [0.48] mmol/l) was obtained (all p values <0.05). There were no adverse side effects. CONCLUSIONS/INTERPRETATION: The unconditioned blood glucose decrease on day 1 and the unconditioned and conditioned increases of peripheral insulin are indicative of brain-pancreas cross-talk. The conditionability of the hormonal responses reveals new applications for intranasal insulin. TRIAL REGISTRATION: DRKS00000537 http://apps.who.int/trialsearch/ FUNDING: Deutsche Forschungsgemeinschaft DFG STO 323/1-1 and 1-2.

[Indicators of course of inpatient therapy in offenders with substance dependency]. / Indikatoren für den Verlauf einer stationären Behandlung bei Straftätern mit Substanzmittelabhängigkeit.

The objective of this review is to summarise the recent state of research on intake criteria for forensic psychiatry in Germany. Therefore, a systematic literature review was conducted on the legal basis of paragraph 64 of the German Penal Code for forensic psychiatry. Although the patients were very heterogeneous, relatively robust indicators were identified that may yield an unsuccessful therapy outcome. A younger age, previous delinquency, absence of an educational and vocational qualification, and personality disorders are the most robust indicators adversely affecting therapy in German forensic psychiatric institutions.

Prothrombin complex concentrate and recombinant prothrombin alone or in combination with recombinant factor X and FVIIa in dilutional coagulopathy: a porcine model.

BACKGROUND: This study was conducted to assess whether newly developed recombinant clotting factor concentrates enable the reversal of dilutional coagulopathy. METHODS: In 50 anesthetized pigs, ~60% of the blood volume was withdrawn and replaced with hydroxyethyl starch. Pigs were randomized to receive either 200 mg kg(-1) fibrinogen (n = 10), fibrinogen and 35 IU kg(-1) prothrombin complex concentrate (PCC) (n = 10), fibrinogen and 4 mg kg(-1) recombinant human factor II (rhFII) concentrate (n = 10), fibrinogen and a three-factor combination (3F) of 4 mg kg(-1) rhFII, 0.006 mg kg(-1) recombinant human FVIIa and 0.32 mg kg(-1) recombinant human FX (n = 10), or saline (n = 10). Thereafter, a standardized liver laceration was performed to induce uncontrolled hemorrhage. Survival time and blood loss were determined, and standard coagulation tests and thrombelastometry were performed. RESULTS: Fibrinogen combined with rhFII or PCC improved survival. Blood loss was significantly decreased in all groups as compared with the animals receiving saline. Clotting time was significantly shortened in the animals treated with fibrinogen and PCC, as well as in those treated with fibrinogen and 3F. One animal died after administration of fibrinogen and PCC. CONCLUSION: Following hemodilution, a combination of fibrinogen and PCC, rhFII or 3F enhances coagulation and final clot strength. Mortality was reduced statistically significantly only in the animals treated with fibrinogen and rhFII or PCC, whereas administration of the combination of fibrinogen and PCC caused a fatal thromboembolic complication. The combination of fibrinogen and rhFII might be effective in reversing dilutional coagulopathy and may reduce blood loss in cases of dilutional coagulopathy.

Role of fibrinogen in trauma-induced coagulopathy.

Coagulation defects related to severe trauma, trauma-induced coagulopathy (TIC), have a number of causal factors including: major blood loss with consumption of clotting factors and platelets, and dilutional coagulopathy after administration of crystalloids and colloids to maintain blood pressure. In addition, activation of the fibrinolytic system or hyperfibrinolysis, hypothermia, acidosis, and metabolic changes can also affect the coagulation system. All of these directly affect fibrinogen polymerization and metabolism. Other bleeding-related deficiencies usually develop later in massive bleeding related to severe multiple trauma. In major blood loss, fibrinogen reaches a critical value earlier than other procoagulatory factors, or platelets. The question of the critical threshold value is presently the subject of heated debate. A threshold of 100 mg dl(-1) has been recommended, but recent clinical data have shown that at a fibrinogen level of <150-200 mg dl(-1), there is already an increased tendency to peri- and postoperative bleeding. A high fibrinogen count exerts a protective effect with regard to the amount of blood loss. In multiple trauma patients, priority must be given to early and effective correction of impaired fibrin polymerization by administering fibrinogen concentrate.