Protamine 1 as a secreted colorectal cancer-specific antigen facilitating G1/S phase transition under nutrient stress conditions.

PURPOSE: Cancer testis antigens (CTAs) are optimal tumor diagnostic markers and involved in carcinogenesis. However, colorectal cancer (CRC) related CTAs are less reported with impressive diagnostic capability or relevance with tumor metabolism rewiring. Herein, we demonstrated CRC-related CTA, Protamine 1 (PRM1), as a promising diagnostic marker and involved in regulation of cellular growth under nutrient deficiency. METHODS: Transcriptomics of five paired CRC tissues was used to screen CRC-related CTAs. Capability of PRM1 to distinguish CRC was studied by detection of clinical samples through enzyme linked immunosorbent assay (ELISA). Cellular functions were investigated in CRC cell lines through in vivo and in vitro assays. RESULTS: By RNA-seq and detection in 824 clinical samples from two centers, PRM1 expression were upregulated in CRC tissues and patients` serum. Serum PRM1 showed impressive accuracy to diagnose CRC from healthy controls and benign gastrointestinal disease patients, particularly more sensitive for early-staged CRC. Furthermore, we reported that when cells were cultured in serum-reduced medium, PRM1 secretion was upregulated, and secreted PRM1 promoted CRC growth in culture and in mice. Additionally, G1/S phase transition of CRC cells was facilitated by PRM1 protein supplementation and overexpression via activation of PI3K/AKT/mTOR pathway in serum deficient medium. CONCLUSIONS: In general, our research presented PRM1 as a specific CRC antigen and illustrated the importance of PRM1 in CRC metabolism rewiring. The new vulnerability of CRC cells was also provided with the potential to be targeted in future. Diagnostic value and grow factor-like biofunction of PRM1 A represents the secretion process of PRM1 regulated by nutrient deficiency. B represents activation of PI3K/AKT/mTOR pathway of secreted PRM1.

Towards antigen-specific Tregs for type 1 diabetes: Construction and functional assessment of pancreatic endocrine marker, HPi2-based chimeric antigen receptor.

Type 1 Diabetes (T1D) is an autoimmune disease marked by direct elimination of insulin-producing ß cells by autoreactive T effectors. Recent T1D clinical trials utilizing autologous Tregs transfers to restore immune balance and improve disease has prompted us to design a novel Tregs-based antigen-specific T1D immunotherapy. We engineered a Chimeric Antigen Receptor (CAR) expressing a single-chain Fv recognizing the human pancreatic endocrine marker, HPi2. Human T cells, transduced with the resultant HPi2-CAR, proliferated and amplified Granzyme B accumulation when co-cultured with human, but not mouse ß cells. Furthermore, following exposure of HPi2-CAR transduced cells to islets, CD8+ lymphocytes demonstrated enhanced CD107a (LAMP-1) expression, while CD4+ cells produced increased levels of IL-2. HPi2-CAR Tregs failed to maintain expansion due to a persistent tonic signaling from the CAR engagement to unexpectantly HPi2 antigen present on Tregs. Overall, we show lack of functionality of HPi2-CAR and highlight the importance of careful selection of CAR recognition driver for the sustainable activity and expandability of engineered T cells.

New monoclonal antibodies specific for mammalian protamines P1 and P2.

The expression of protamines and the binding of these small arginine-rich proteins to DNA complete the process of spermatid chromatin reorganization and the global inactivation of the male's haploid genome that occurs during the final stages of sperm development in mammals. While a number of anti-protamine antibodies have been created during the last 40 years, only a few have proven useful for detecting the presence of the protamines, determining the timing of their expression and deposition in chromatin, and investigating their structure and function in both maturing spermatids and sperm. The aim of this effort was to develop an additional set of monoclonal antibodies (MAbs) that not only recognize new P1 and P2 protamine epitopes but also work well as IHC reagents for detecting and identifying mammalian protamines in testicular tissue and ejaculated sperm. Using a combination of native and synthetic human protamines as antigens, 38 hybridoma clones recognizing human protamine P1 or P2 were generated. Antibodies produced by the 12 best clones were screened for selectivity by enzyme-linked immunosorbent assay, and two were found to recognize only human protamine P1 or P2, while a number of the others bound to both the human and mouse proteins. One MAb recognized every protamine tested. All the antibodies, including one recognizing stallion P1 and another recognizing stallion P2, bound to the native protamines in the chromatin of spermatids or sperm. While the majority labeled only elongating spermatids or sperm, several of the antibodies were found to also bind to the cytoplasm or nuclei of cells that lack protamine, which indicates these MAbs must recognize epitopes present in the protamines that are also found in other proteins. Thirteen overlapping human protamine P1 peptides were synthesized and subsequently used to identify the epitopes recognized by the six best antibodies. Abbreviations: BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; HCl: hydrochloric acid; IHC: immunohistochemistry; i.p: intraperitoneal; LIS: lithium diiodosalicylate; MAb: monoclonal antibody; PBS: phosphate buffered saline.

Polymeric Nanocapsules for Vaccine Delivery: Influence of the Polymeric Shell on the Interaction With the Immune System.

The use of biomaterials and nanosystems in antigen delivery has played a major role in the development of novel vaccine formulations in the last few decades. In an effort to gain a deeper understanding of the interactions between these systems and immunocompetent cells, we describe here a systematic in vitro and in vivo study on three types of polymeric nanocapsules (NCs). These carriers, which contained protamine (PR), polyarginine (PARG), or chitosan (CS) in the external shell, and their corresponding nanoemulsion were prepared, and their main physicochemical properties were characterized. The particles had a mean particle size in the range 250-450 nm and a positive zeta potential (~30-40 mV). The interaction of the nanosystems with different components of the immune system were investigated by measuring cellular uptake, reactive oxygen species production, activation of the complement cascade, cytokine secretion profile, and MAP kinases/nuclear factor κB activation. The results of these in vitro cell experiments showed that the NC formulations that included the arginine-rich polymers (PR and PARG) showed a superior ability to trigger different immune processes. Considering this finding, protamine and polyarginine nanocapsules (PR and PARG NCs) were selected to assess the association of the recombinant hepatitis B surface antigen (rHBsAg) as a model antigen to evaluate their ability to produce a protective immune response in mice. In this case, the results showed that PR NCs elicited higher IgG levels than PARG NCs and that this IgG response was a combination of anti-rHBsAg IgG1/IgG2a. This work highlights the potential of PR NCs for antigen delivery as an alternative to other positively charged nanocarriers.

Harnessing RNA sequencing for global, unbiased evaluation of two new adjuvants for dendritic-cell immunotherapy.

Effective stimulation of immune cells is crucial for the success of cancer immunotherapies. Current approaches to evaluate the efficiency of stimuli are mainly defined by known flow cytometry-based cell activation or cell maturation markers. This method however does not give a complete overview of the achieved activation state and may leave important side effects unnoticed. Here, we used an unbiased RNA sequencing (RNA-seq)-based approach to compare the capacity of four clinical-grade dendritic cell (DC) activation stimuli used to prepare DC-vaccines composed of various types of DC subsets; the already clinically applied GM-CSF and Frühsommer meningoencephalitis (FSME) prophylactic vaccine and the novel clinical grade adjuvants protamine-RNA complexes (pRNA) and CpG-P. We found that GM-CSF and pRNA had similar effects on their target cells, whereas pRNA and CpG-P induced stronger type I interferon (IFN) expression than FSME. In general, the pathways most affected by all stimuli were related to immune activity and cell migration. GM-CSF stimulation, however, also induced a significant increase of genes related to nonsense-mediated decay, indicating a possible deleterious effect of this stimulus. Taken together, the two novel stimuli appear to be promising alternatives. Our study demonstrates how RNA-seq based investigation of changes in a large number of genes and gene groups can be exploited for fast and unbiased, global evaluation of clinical-grade stimuli, as opposed to the general limited evaluation of a pre-specified set of genes, by which one might miss important biological effects that are detrimental for vaccine efficacy.

Generation of Immunostimulating 130 nm Protamine-RNA nanoparticles.

Nanoparticles of defined size can be easily obtained by simply mixing Protamine, a pharmaceutical drug that is used to neutralize heparin after surgery, and RNA in the form of oligonucleotides or messenger RNA. Depending on the concentrations of the two reagents and their salt contents, homogenous nanoparticles with a mean diameter of 50 to more than 1000 nm can spontaneously be generated. RNA is a danger signal because it is an agonist of for example TLR-3, -7, and -8; therefore, Protamine-RNA nanoparticles are immunostimulating. We and others have shown in vitro that nanoparticle size and interferon-alpha production by human peripheral blood mononuclear cells (PBMCs) are inversely correlated. Conversely, nanoparticle size and TNF-alpha production by PBMCs are positively correlated (Rettig et al., Blood 115:4533-4541, 2010). Particles of less than 450 nm are most frequently used for research and clinical applications because they are very stable, remain polydispersed and induce interferon-alpha proteins, which are a natural antiviral and anticancer protein family with 12 members in humans. Herein, we describe a method to generate 130 nm nanoparticles as well as some of their physical and biological characteristics.

Purification and Characterization of Protamine, the Allergen from the Milt of Large Yellow Croaker (Pseudosciaena crocea), and Its Components.

The protamine in fish milt can cause anaphylaxis in humans. To determine the allergen in the milt of large yellow croaker (Pseudosciaena crocea), crude extracts were incubated with sera from allergic patients. The results showed that a 12 kDa multicomponent protein was the major allergen in the milt of large yellow croaker. The multicomponent protein was purified, and physicochemical characterization showed that it was a glycoprotein, highly stable in acid-alkali conditions, and weakly retained immunoglobulin E (IgE)-binding activity at high temperatures. Separation and immunoreactivity analysis of the components of the multicomponent protein showed that it had six components, and component 5 had the strongest IgE-binding activity with patient sera. N-terminal sequencing confirmed the multicomponent protein was protamine. Following analysis of protamine from different fish by reversed-phase liquid chromatography and circular dichroism spectra, the protamines from different fish were found to have a similar secondary structure, although their components were different.

Partially desulfated heparin modulates the interaction between anti-protamine/heparin antibodies and platelets.

Protamine (PRT) is the standard drug to neutralise heparin. PRT/heparin complexes induce an immune response similar to that observed in heparin-induced thrombocytopenia (HIT). Partially desulfated heparin (ODSH) was shown to interfere with anti-platelet factor 4/heparin antibodies (Abs), which are responsible for HIT. In this study, we analyse the impact of ODSH on the interaction between anti-PRT/heparin Abs and platelets. The ability of ODSH to prevent anti-PRT/heparin Ab-induced platelet destruction in vivo was investigated using the NOD/SCID mouse model. ODSH improved platelet survival in the presence of PRT, heparin and anti-PRT/heparin Abs (median platelet survival after 300 minutes (min) with 20 µg/ml ODSH: 75%, range 70-81% vs without ODSH: 49%, range 44-59%, p=0.006). Furthermore, when ODSH was applied 60 min after Ab injection platelet survival was improved (median platelet survival after 300 min with ODSH: 83%, range 77-93% vs without ODSH: 59%, range 29-61%, p=0.02). In in vitro experiments ODSH inhibited platelet activation at concentrations >16 µg/mL (p<0.001), as well as PRT/heparin complex binding to platelets (mean fluorescence intensity [MFI] without ODSH: 85 ± 14 vs with ODSH: 15 ± 0.6, p=0.013). ODSH also displaced pre-bound complexes from the platelet surface (MFI without ODSH: 324 ± 43 vs with 32 µg/ml ODSH: 53 ± 9, p<0.001). While interfering with platelet activation by anti-PRT/heparin Abs, up to a concentration of 16 µg/ml, ODSH had only minimal impact on neutralisation of heparin by PRT. In conclusion, our study shows that ODSH is able to inhibit platelet activation and destruction suggesting a potential clinical use to reduce anti-PRT/heparin Ab-mediated adverse effects.

A commercial human protamine-2 antibody used in several studies to detect mouse protamine-2 recognizes mouse transition protein-2 but not protamine-2.

The exchange of histones for transition proteins (TNPs) and finally protamines is an essential process during spermatogenesis that enables the strong condensation of chromatin during sperm formation. Research on this process obviously depends on the availability of specific antibodies recognizing these nuclear proteins. A commercial antibody generated against human protamine-2 (PRM2) has been described to cross-react with mouse PRM2 and in fact has been used in several studies to detect mouse PRM2. Some inconsistent results obtained with this goat-derived antibody prompted us to re-examine its specificity. In immunofluorescence experiments with epididymal sperm, only a low percentage of sperm nuclei were stained by this antibody, whereas a mouse monoclonal anti- PRM2 antibody stained most sperm, as expected. Western blot analysis of basic nuclear proteins from spermatids and sperm separated by acid urea (AU) gel electrophoresis revealed that the goat anti- PRM2 antiserum binds to mouse TNP2 but not mouse PRM2. Epitope mapping using glutathione-S-transferase-fusion proteins with peptide sequences conserved in human PRM2 and mouse TNP2 identified the tetrapeptide arginyl-lysyl-arginyl-threonine as an epitope of the goat anti- PRM2 antiserum. Our findings underline the importance of using AU gel electrophoresis to confirm specificities of antibodies directed against basic nuclear proteins, which are not well separated, and may show abnormal migration behaviour, in SDS-polyacrylamide gel electrophoresis.

Platelet-activating protamine-heparin-antibodies lead to higher protamine demand in patients undergoing cardiac surgery.

OBJECTIVES: Platelet-activating antibodies against protamine-heparin-complexes were described in patients undergoing cardiac surgery, but their clinical consequences remain unclear. This prospective single-center observational study aimed to describe the prevalence and clinical consequences of protamine-heparin-complex antibodies in patients undergoing cardiac surgery with cardiopulmonary bypass. METHODS: A total of 200 patients undergoing cardiac surgery with cardiopulmonary bypass were included. Blood samples were collected preoperatively and 1 hour, 24 hours, and 7 days after weaning from cardiopulmonary bypass. All sera were tested for the presence of protamine-heparin-complex antibodies using a modified heparin-induced platelet-activation assay. Specific Fcγ receptor IIa-dependent platelet activation was confirmed by repeated testing in the presence of the Fcγ receptor IIa-blocking antibody IV.3. RESULTS: Samples from 185 patients were obtained, of whom 24 patients (13%) were positive for protamine-heparin-complex antibodies preoperatively. In all positive samples, functional reactivity was reversible in the presence of IV.3. Although patients with a preoperative presence of protamine-heparin-complex antibodies were significantly older compared with patients negative for protamine-heparin-complex antibodies (73 ± 9.8 years vs 68 ± 10 years, P = .037), no other potential risk factors were identified at 1 day before operation. Patients with protamine-heparin-complex antibodies required significantly more protamine to neutralize heparin (47.66 mg vs 41.67 mg, P = .027). Protamine-heparin-complex antibodies have no significant influence on perioperative platelet numbers, bleeding complications, transfusion requirement, thromboembolic events, major cardiovascular and cerebrovascular events, inflammation parameters, or kidney function. CONCLUSIONS: Protamine-heparin-complex antibodies occur frequently in patients undergoing cardiac surgery on cardiopulmonary bypass, resulting in specific platelet activation in vitro. Protamine-heparin-complex antibodies are associated with increased protamine requirement after cardiopulmonary bypass and possibly slower recovery of platelet numbers.

Serological features of antibodies to protamine inducing thrombocytopenia and thrombosis.

BACKGROUND: A significant proportion of patients undergoing cardiopulmonary bypass develop anti-protamine antibodies, with or without the association of thromboembolic events. METHODS: We extensively investigated the serological features of protamine antibodies, which developed in six patients who were clinically suspected to have heparin-induced thrombocytopenia (HIT). Three patients had thrombotic events. Sera were tested by four different commercially available immunoassays, a heparin-platelet aggregation test, and for their binding properties to heparin, platelet factor 4 (PF4), complex heparin-PF4, protamine, and protamine complex with heparin. Sera from four patients were also tested for the capability to induce platelet activation and the formation of platelet-monocyte heterotypic aggregates. RESULTS: The ELISA assay Zymutest HIA was strongly positive in all cases, the HPIA Asserachrome was borderline, and the gel centrifugation test PaDGIA was positive in two tested patients. Platelet aggregation tests were negative. Using a variation of the Zymutest HIA we demonstrate that IgG antibodies bound only to protamine or protamine complex with heparin, but not to heparin or PF4 only. Sera-induced platelet P-selectin expression and the formation of platelet-monocyte aggregates. Blood samples from one patient proofed positive concomitantly with the thromboembolic event. However, serological characteristics did not differ between antibodies associated with thromboembolic events from those without. CONCLUSIONS: These data show that protamine-induced antibodies are specific and may induce platelet activation, which explains their association with thromboembolic events.

Low molecular weight protamine (LMWP): a nontoxic protamine substitute and an effective cell-penetrating peptide.

Low molecular weight protamine (LMWP) is a peptide fragment produced in our laboratory from enzymatic digestion of native protamine. More than 30 papers studying the properties and applications of LMWP have been published by our group in various journals since its initial discovery in 1999. Results have shown that LMWP could completely neutralize the anticoagulant functions of both heparin and low molecular weight heparin (LMWH), with reduced antigenicity and cross-reactivity toward the mice-derived anti-protamine antibodies. Aside from its potential as a heparin/LMWH antagonist, LMWP also shows the ability to retard insulin adsorption by the formation of an insoluble complex, making it a less toxic long-lasting insulin product than the conventional neutral protamine Hagedorn (NPH) insulin for diabetic control. Importantly, LMWP (Sequence: VSRRRRRRGGRRRR), with 10 arginine residues in its structure, could function as a cell-penetrating peptide (CPP), also termed protein transduction domain (PTD), to achieve effective intracellular protein or gene delivery in clinical practice. In this paper, we present a thorough review of our work related to LMWP, with the aim of providing readers an insight into its potential to be a clinical protamine substitute as well as a non-toxic cell penetrating peptide applicable to achieve intracellular protein and gene delivery.

Protamine-containing insulin but not analog insulin and duration of insulin use are risk factors for the production of insulin autoantibodies in insulin-treated patients with diabetes mellitus.

Insulin autoantibodies can be produced by insulin injections but rarely cause severe side effects such as glucose instability and insulin allergy. We study the characteristics of insulin autoantibody-positive diabetic patients with a medical history of insulin therapy using single and multiple (adjusted for age, sex, type of diabetes) logistic regression analyses. Associations between insulin autoantibodies and age, sex, type of diabetes, HbA1c, and serum creatinine were not significant, but the association between insulin autoantibodies and duration of insulin use was significant. Unadjusted and adjusted odds ratios were 1.08 (1.02-1.14) and 1.07 (1.01-1.14), respectively. Unadjusted and adjusted odds ratios for protamine-containing insulin were 3.08 (1.49-6.34) and 4.27 (1.90-9.58), respectively. The adjusted odds ratios for premixed biphasic insulin and intermediate-acting insulin were 2.21 (1.03-4.73) and 2.35 (1.01-5.49), respectively. Associations between insulin autoantibodies and any insulin analog were not significant. These results suggest that protamine-containing insulin and duration of insulin use are risk factors for the production of insulin autoantibodies. If patients with poorly controlled diabetes have a history of protamine-containing insulin therapy over a long time, the appearance of insulin autoantibodies should be monitored.

Incidence of antibodies to protamine sulfate/heparin complexes incardiac surgery patients and impact on platelet activation and clinical outcome.

A new ELISA (Zymutest HIA®), based on incubation of diluted plasma with protamine/heparin (PRT/H) complexes without and with platelet factor 4 (PF4) provided by a platelet lysate, was used to detect heparin-dependent antibodies in a cohort of 232 cardiac surgery (CS) patients and in 47 patients with heparin-induced thrombocytopenia (HIT). Significant binding of IgG/A/M to PRT/H complexes was demonstrated in 59 CS patients (25.4%), with similar absorbances whether platelet lysate was added to the plasma or not, and significant reactivity to PF4/H in 29 of them. Antibodies to PRT or heparin alone were present in 15 and two of these patients, respectively. Importantly, antibodies to PRT/H were detected in only three of the 47 HIT patients, who had also undergone recent CS. The Zymutest HIA® was positive in another 41 CS patients (17%), but only or mainly when their plasma was tested with platelet lysate, with significant levels of antibodies to PF4/H in 40 of them without detectable reactivity to PRT or heparin alone. Slight antibody binding to PRT/H complexes was also measured in six of these 41 patients. Therefore, a total of 35 CS patients exhibited dual antibody reactivity towards PRT/H and PF4/H complexes. Serotonin release assay performed with PRT alone was positive in 17 CS patients with antibodies to PRT/H, but all had normal platelet count evolution without thrombosis postoperatively. In conclusion, antibodies to PRT/H are frequently present in CS patients postoperatively (25.4%), and can activate platelets in vitro, but their clinical impact remains questionable.

Protamine-induced thrombocytopenia?

In this issue of Blood, Bakchoul et al and Lee et al describe and characterize a common but only recently recognized immune response to protamine after cardiopulmonary bypass (CPB) surgery with potential important clinical implications.

Protamine-induced immune thrombocytopenia.

BACKGROUND: Protamine is widely used to reverse the anticoagulant effects of heparin. Although mild thrombocytopenia is common in patients given protamine after cardiac procedures, acute severe thrombocytopenia has not been described. We encountered a patient who experienced profound thrombocytopenia and bleeding shortly after administration of protamine and performed studies to characterize the responsible mechanism. STUDY DESIGN AND METHODS: Patient serum was studied for antibodies that recognize protamine, heparin-protamine complexes, and platelets (PLTs) treated with protamine using flow cytometry, enzyme-linked immunosorbent assay, and serotonin release from labeled PLTs. RESULTS: A high-titer immunoglobulin G antibody was detected in patient serum that recognizes protamine in a complex with heparin or PLT surface glycosaminoglycans (GAGs) and activates PLTs treated with protamine at concentrations achieved in vivo after protamine infusion. The antibody is distinctly different from those found in patients with heparin-induced thrombocytopenia on the basis of its failure to recognize heparin in a complex with PLT factor 4 (PF4) and to release serotonin from labeled PLTs in the absence of protamine. CONCLUSIONS: Findings made suggest that the patient's antibody is specific for conformational changes induced in protamine when it reacts with heparin or a PLT surface GAG. Development of severe thrombocytopenia after treatment of this patient with protamine defines a previously undescribed mechanism of drug-induced immune thrombocytopenia. Patients given protamine who produce this type of antibody may be at risk of experiencing thrombocytopenia if given the drug a second time while antibody is still present.

High incidence of antibodies to protamine and protamine/heparin complexes in patients undergoing cardiopulmonary bypass.

Protamine is routinely used to reverse heparin anticoagulation during cardiopulmonary bypass (CPB). Heparin interacts with protamine to form ultralarge complexes that are immunogenic in mice. We hypothesized that patients exposed to protamine and heparin during CPB will develop antibodies (Abs) to protamine/heparin (PRT/H) complexes that are capable of platelet activation. Specimens from a recently completed prospective clinical trial (HIT [for heparin-induced thrombocytopenia] 5801 study; n = 500) of CPB patients were examined for PRT/H Abs at baseline, at time of hospital discharge (between days 3 through 7), and 30 days after CPB. PRT/H antibody features were characterized and correlated with adverse cardiovascular outcomes. We found a high incidence of PRT/H antibody formation (29%) in patients undergoing cardiac surgery. PRT/H Abs were of high titer (mean titer 1:14,744), showed heparin-dependent binding, and activated platelets in the presence of protamine. PRT/H Abs showed no cross-reactivity to platelet factor 4/heparin complexes, but were cross-reactive with protamine-containing insulin preparations. In the absence of circulating antigen at day 30, there were no complications of thrombocytopenia, thrombotic events, or long-term cardiovascular events. These studies show that Abs to PRT/H occur commonly after cardiac bypass surgery, share a number of serologic features with HIT Abs, including platelet activation, and may pose health risks to patients requiring drug reexposure.

Anti-protamine-heparin antibodies: incidence, clinical relevance, and pathogenesis.

Protamine, which is routinely used after cardiac surgery to reverse the anticoagulant effects of heparin, is known to be immunogenic. Observing patients with an otherwise unexplained rapid decrease in platelet count directly after protamine administration, we determined the incidence and clinical relevance of protamine-reactive antibodies in patients undergoing cardiac-surgery. In vitro, these antibodies activated washed platelets in a FcγRIIa-dependent fashion. Using a nonobese diabetic/severe combined immunodeficiency mouse model, those antibodies induced thrombocytopenia only when protamine and heparin were present but not with protamine alone. Of 591 patients undergoing cardiopulmonary bypass surgery, 57 (9.6%) tested positive for anti-protamine-heparin antibodies at baseline and 154 (26.6%) tested positive at day 10. Diabetes was identified as a risk factor for the development of anti-protamine-heparin antibodies. In the majority of the patients, these antibodies were transient and titers decreased substantially after 4 months (P < .001). Seven patients had platelet-activating, anti-protamine-heparin antibodies at baseline and showed a greater and more prolonged decline in platelet counts compared with antibody-negative patients (P = .003). In addition, 2 of those patients experienced early arterial thromboembolic complications vs 9 of 584 control patients (multivariate analysis: odds ratio, 21.58; 95% confidence interval, 2.90-160.89; P = .003). Platelet-activating anti-protamine-heparin antibodies show several similarities with anti-platelet factor 4-heparin antibodies and are a potential risk factor for early postoperative thrombosis.