Cardiac surgery in acute heparin-induced thrombocytopenia managed with therapeutic plasma exchange and intravenous immunoglobulin.

Background: Urgent surgery requiring heparin exposure during cardiopulmonary bypass can be challenging in patients with acute heparin-induced thrombocytopenia (HIT). The use of treatments such as therapeutic plasma exchange (TPE) to remove HIT antibodies and intravenous immunoglobulin (IVIg) to antagonize HIT antibody-mediated platelet activation are increasingly reported in patients who undergo cardiac surgery. The optimal treatment approach to mitigate the risks of heparin administration in this situation is not known. Key Clinical Question: Can TPE coupled to IVIg allow for safe heparin exposure in patients with HIT? Clinical Approach: TPE and IVIg were used to enable heparin exposure for surgical placement of a left ventricular assist device in a patient with HIT. Serial patient samples were tested in antigen-based and functional HIT assays. Conclusion: Dissociation between antigen-based (enzyme-linked immunosorbent assay) and functional (serotonin release assay) testing was noted, and TPE coupled to IVIg was associated with an excellent clinical response.

Cerebral venous sinus thrombosis associated with SRA-negative heparin-induced thrombocytopenia: case report.

BACKGROUND: There are very few documented reports in literature of cerebral venous sinus thrombosis (CVST) caused by immune-mediated heparin-induced thrombocytopenia (HIT). Further, there are very few reports of false negative serotonin release assays (SRAs) when testing for immune-mediated HIT. CASE PRESENTATION: We present a case of a 60- year-old male with recent unfractionated heparin administration for venous thromboembolism prophylaxis, an elevated 4T score of 5 and acute CVST in which immune-mediated HIT was suspected. The enzyme-linked immunosorbent assay (ELISA) screening assay was positive for PF4 antibodies and subsequent reflexive SRA testing was negative. However, given the clinical picture, a false-negative SRA was suspected (and eventually confirmed), prompting use of the alternative PF4-dependent p-selectin expression assay (PEA) which was confirmed to be positive. The patient was successfully managed with a bivalirudin infusion and eventually transitioned to apixaban. CONCLUSION: It is uncommon for immune-mediated HIT with thrombosis to manifest as CVST. Similarly, false-negative SRA is uncommon in immune-mediated HIT. Take-away lessons from our case report include considering HIT in CVST patients with an elevated 4T score and considering the entire clinical picture and degree of suspicion for HIT when interpreting negative HIT testing results. The PEA, in conjunction with the 4Ts score, may be considered as an alternate diagnostic assay for HIT.

Venous thromboembolism after COVID-19 vaccination in patients with thrombophilia.

Patients with thrombophilia remain concerned about venous thromboembolism (VTE) risk with COVID-19 vaccinations. The aim of this study was to examine VTE outcomes in patients with inherited or acquired thrombophilia who were vaccinated for COVID-19. Vaccinated patients ≥18 years between November 1, 2020 and November 1, 2021 were analyzed using electronic medical records across the Mayo Clinic enterprise. The primary outcome was imaging confirmed acute VTE occurring 90 days before and after the date of the first vaccine dose. Thrombophilia patients were identified through laboratory testing results and ICD-10 codes. A total of 792 010 patients with at least one COVID-19 vaccination were identified. Six thousand sixty-seven of these patients were found to have a thrombophilia, among whom there was a total of 39 VTE events after compared to 51 VTE events before vaccination (0.64% vs. 0.84%, p = .20). In patients with Factor V Leiden or prothrombin gene mutation, VTE occurred in 27 patients before and in 29 patients after vaccination (0.61 vs. 0.65%, p = .79). In patients with antiphospholipid syndrome, VTE occurred in six patients before and four patients after vaccination (0.59% vs. 0.39%, p = .40). No difference was observed in the overall VTE rate when comparing the postvaccination 90 days to the prevaccination 90 days, adjusted hazard ratio 0.81 (95% confidence interval: 0.53-1.23). In this subgroup of COVID-19 vaccinated patients with thrombophilia, there was no increased risk for acute VTE postvaccination compared to the prevaccination timeframe. These results are consistent with prior studies and should offer additional reassurance to patients with inherited or acquired thrombophilia.

Cloned antibodies from patients with HIT provide new clues to HIT pathogenesis.

Heparin-induced thrombocytopenia (HIT) is a serious adverse drug reaction characterized by antibodies that recognize platelet factor 4/heparin complexes (PF4/H) and activate platelets to create a prothrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset also makes antibodies that are platelet activating (PA). A close correlation between PA antibodies and the likelihood of experiencing HIT has been demonstrated in clinical studies, but how PA (presumptively pathogenic) and nonactivating (NA) (presumptively benign) antibodies differ from each other at the molecular level is unknown. To address this issue, we cloned 7 PA and 47 NA PF4/H-binding antibodies from 6 patients with HIT and characterized their structural and functional properties. Findings showed that PA clones differed significantly from NA clones in possessing 1 of 2 heavy chain complementarity-determining region 3 (HCDR3) motifs, RX1-2R/KX1-2R/H (RKH) and YYYYY (Y5), in an unusually long complementarity-determining region 3 (≥20 residues). Mutagenic studies showed that modification of either motif in PA clones reduced or abolished their PA activity and that appropriate amino acid substitutions in HCDR3 of NA clones can cause them to become PA. Repertoire sequencing showed that the frequency of peripheral blood IgG+ B cells possessing RKH or Y5 was significantly higher in patients with HIT than in patients without HIT given heparin, indicating expansion of B cells possessing RKH or Y5 in HIT. These findings imply that antibodies possessing RKH or Y5 are relevant to HIT pathogenesis and suggest new approaches to diagnosis and treatment of this condition.

Vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is primarily a complication of adenoviral vector-based covid-19 vaccination. In VITT, thrombocytopenia and thrombosis mediated by anti-platelet factor 4 (PF4) antibodies can be severe, often characterized by thrombosis at unusual sites such as the cerebral venous sinus and splanchnic circulation. Like in heparin-induced thrombocytopenia (HIT) and spontaneous HIT, VITT antibodies recognize PF4-polyanion complexes and activate PF4-treated platelets but additionally bind to un-complexed PF4, a critical finding that could be leveraged for more specific detection of VITT. Intravenous immunoglobulin and non-heparin-based anticoagulation remain the mainstay of treatment. Second dose/boosters of mRNA covid-19 vaccines appear safe in patients with adenoviral vector-associated VITT. Emerging data is consistent with the possibility that ultra-rare cases of VITT may be seen in the setting of mRNA and virus-like particle (VLP) technology-based vaccinations and until more data is available, it is prudent to consider VITT in the differential diagnosis of all post-vaccine thrombosis and thrombocytopenia reactions.

Off-the-shelf cryopreserved platelets for the detection of HIT and VITT antibodies.

Heparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis.

Risk of venous thromboembolism after COVID-19 vaccination.

BACKGROUND: COVID-19 vaccinations in the United States are effective in preventing illness and hospitalization yet concern over post-vaccination venous thromboembolism (VTE) risk has led to vaccine hesitancy. METHODS: The aim of this study was to compare VTE rates before and after COVID-19 vaccination. COVID-19 vaccinated patients ≥18 years between November 1, 2020 through November 1, 2021 were analyzed using electronic medical records across the Mayo Clinic enterprise. The primary outcome was imaging confirmed acute VTE (upper or lower deep vein thrombosis or pulmonary embolism) occurring 90 days before and after the date of first vaccine dose. RESULTS: A total of 792 010 patients with at least one COVID-19 vaccination were identified (Pfizer, n = 452 950, Moderna, n = 290 607, and Janssen [Johnson & Johnson], n = 48 453). A total of 1565 VTE events occurred in the 90 days before (n = 772) and after (n = 793) COVID-19 vaccination. VTE post-vaccination occurred in 326 patients receiving Moderna (0.11%, incidence rate [IR] 4.58 per 1000p-years), 425 patients receiving Pfizer (0.09%, IR 3.84 per 1000p-years), and 42 receiving Janssen (0.09%, IR 3.56 per 1000p-years). Compared to the pre-vaccination timeframe, the adjusted hazard ratio (aHR) for VTE after the Janssen vaccination was 0.97 (95% confidence interval [CI] 0.63-1.50), aHR 1.02 (95% CI 0.87-1.19) for Moderna, and aHR 1.00 (95% CI 0.87-1.15) for Pfizer. CONCLUSION: In this large cohort of COVID-19 vaccinated patients, no increased risk for acute VTE post-vaccination was identified for the authorized vaccines in the United States.

Chemoenzymatic Synthesis of Homogeneous Heparan Sulfate and Chondroitin Sulfate Chimeras.

Heparan sulfate (HS) and chondroitin sulfate (CS) are two structurally distinct natural polysaccharides. Here, we report the synthesis of a library of seven structurally homogeneous HS and CS chimeric dodecasaccharides (12-mers). The synthesis was accomplished using six HS biosynthetic enzymes and four CS biosynthetic enzymes. The chimeras contain a CS domain on the reducing end and a HS domain on the nonreducing end. The synthesized chimeras display anticoagulant activity as measured by both in vitro and ex vivo experiments. Furthermore, the anticoagulant activity of H/C 12-mer 5 is reversible by protamine, a U.S. Food and Drug Administration-approved polypeptide to neutralize anticoagulant drug heparin. Our findings demonstrate the synthesis of unnatural HS-CS chimeric oligosaccharides using natural biosynthetic enzymes, offering a new class of glycan molecules for biological research.

Persistence of Ad26.COV2.S-associated vaccine-induced immune thrombotic thrombocytopenia (VITT) and specific detection of VITT antibodies.

Rare cases of COVID-19 vaccinated individuals develop anti-platelet factor 4 (PF4) antibodies that cause thrombocytopenia and thrombotic complications, a syndrome referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). Currently, information on the characteristics and persistence of anti-PF4 antibodies that cause VITT after Ad26.COV2.S vaccination is limited, and available diagnostic assays fail to differentiate Ad26.COV2.S and ChAdOx1 nCoV-19-associated VITT from similar clinical disorders, namely heparin-induced thrombocytopenia (HIT) and spontaneous HIT. Here we demonstrate that while Ad26.COV2.S-associated VITT patients are uniformly strongly positive in PF4-polyanion enzyme-linked immunosorbent assays (ELISAs); they are frequently negative in the serotonin release assay (SRA). The PF4-dependent p-selectin expression assay (PEA) that uses platelets treated with PF4 rather than heparin consistently diagnosed Ad26.COV2.S-associated VITT. Most Ad26.COV2.S-associated VITT antibodies persisted for >5 months in PF4-polyanion ELISAs, while the PEA became negative earlier. Two patients had otherwise unexplained mild persistent thrombocytopenia (140-150 x 103 /µL) 6 months after acute presentation. From an epidemiological perspective, differentiating VITT from spontaneous HIT, another entity that develops in the absence of proximate heparin exposure, and HIT is important, but currently available PF4-polyanion ELISAs and functional assay are non-specific and detect all three conditions. Here, we report that a novel un-complexed PF4 ELISA specifically differentiates VITT, secondary to both Ad26.COV2.S and ChAdOx1 nCoV-19, from both spontaneous HIT, HIT and commonly-encountered HIT-suspected patients who are PF4/polyanion ELISA-positive but negative in functional assays. In summary, Ad26.COV2.S-associated VITT antibodies are persistent, and the un-complexed PF4 ELISA appears to be both sensitive and specific for VITT diagnosis.

P-selectin expression assay in a repeatedly serotonin-release assay-negative patient with heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy caused by antibodies to complexes of platelet factor 4 (PF4) and heparin. Pathogenic antibodies to PF4/heparin bind and activate platelets to propagate a hypercoagulable state culminating in life-threatening thrombosis. The serotonin-release assay (SRA) is considered the gold-standard test to diagnose HIT. However, the sensitivity of the SRA was questioned with reported cases of clinical diagnosis of HIT and negative SRA. Herein, we present the utility of platelet factor 4-dependent P-selectin expression assay (PEA) in diagnosing HIT in a patient with thrombocytopenia and recurrent thrombosis who repeatedly tested negative with SRA.

Cerebral venous sinus thrombosis associated with spontaneous heparin-induced thrombocytopenia syndrome after total knee arthroplasty.

Spontaneous heparin-induced thrombocytopenia (HIT) syndrome, characterized by clinical and serologic features of HIT despite the absence of proximate heparin exposure, can be triggered by total knee arthroplasty (TKA). A 56-year-old female receiving aspirin thromboprophylaxis post-TKA presented with aphasia and thrombocytopenia on post-operative day 11. Imaging studies revealed cerebral venous sinus thrombosis (CVST) and intravenous bivalirudin was initiated. Her serum tested strong-positive for IgG anti-PF4/polyanion complexes and serotonin-release assay in the presence and absence of heparin; strong-positive IgG-specific chemiluminescent immunoassay; and moderate-positive latex immunoturbidimetric assay. Two 65 g doses of IVIG were administered. With the improvement of her platelet count, she was transitioned from bivalirudin to warfarin. At one-year follow-up, she remained free of recurrent thrombosis and neurologically stable with a normal platelet count. Previous reports of post-TKA spontaneous HIT syndrome include venous/arterial thrombosis and adrenal hemorrhage, and this report of CVST expands the clinical spectrum of this rare complication of orthopedic surgery.

Heparin-Induced Thrombocytopenia: A Focus on Thrombosis.

Heparin-induced thrombocytopenia is an immune-mediated disorder caused by antibodies that recognize complexes of platelet factor 4 and heparin. Thrombosis is a central and unpredictable feature of this syndrome. Despite optimal management, disease morbidity and mortality from thrombosis remain high. The hypercoagulable state in heparin-induced thrombocytopenia is biologically distinct from other thrombophilic disorders in that clinical complications are directly attributable to circulating ultra-large immune complexes. In some individuals, ultra-large immune complexes elicit unchecked cellular procoagulant responses that culminate in thrombosis. To date, the clinical and biologic risk factors associated with thrombotic risk in heparin-induced thrombocytopenia remain elusive. This review will summarize our current understanding of thrombosis in heparin-induced thrombocytopenia with attention to its clinical features, cellular mechanisms, and its management.