The discovery of heparin was a serendipitous finding by a young medical student, Jay McLean. After applying to Johns Hopkins Medical School and being accepted for the Class of 1916, McLean traveled by train from San Francisco to Baltimore. Upon his arrival, he was told the matriculating class had already started their studies and he would have to wait until the following year’s class to begin his medical school experience.

Consequently, McLean looked for a position in research at Johns Hopkins University. Professor William H. Howell offered him the opportunity to work on the extraction of “thromboplastin” from animal livers. Paradoxically, McLean’s work identified an “anticoagulant substance” rather than a procoagulant substance. Howell and McLean introduced the term “heparin” for the anticoagulant substance extracted from animal livers.

Table 1: History of Heparin

	1916 Jay McLean, William Howell: “Discovery of Heparin”
	1937 C. Crafoord: Heparin prevented thrombosis
	1957 R.E. Weismann, Richard Tobin: peripheral arterial embolism when re-exposed to heparin
	1964 Brooke Roberts & colleagues suggested an antigen-antibody mechanism to explain heparin-induced thrombotic complications.
	1973 Donald Silver and two surgical residents: Glen R. Rhodes and R. H. Dixon: identified triad of clinical findings (1) thrombocytopenia, (2) thrombosis, (3) heparin dependent antibodies.
	1979 Johnathan Towne, a vascular surgeon from Milwaukee, described “white clot syndrome”. His group also described phlegmasia cerulea dolens which progressed to venous limb gangrene.

In 1973, Donald Silver, Glen Rhodes and R.H. Dixon were the first to suggest a heparin-dependent antibody as the cause of thrombocytopenia and complicating thrombosis in vascular surgery patients. (Table 2)

The first two patients described by Professor Silver’s group presented with significant thrombocytopenia, myocardial infarction and petechiae. They also observed the platelet count quickly returned to normal reference intervals upon discontinuing heparin. This group also described recurrence of thrombocytopenia when patients were re-exposed to heparin. When patient serum and heparin were added to normal donor platelets, platelet aggregation was induced. Additional studies identified the aggregating agent as IgG in patient sera.

Table 2: HIT: Pathogenesis*

• Rhodes et al. established immune basis of thrombocytopenia.

• Adding patient serum to normal donor platelets in the presence of heparin resulted in platelet aggregation.

• Aggregation of platelets was induced by IgG from patients serum in the presence of heparin.

* Rhodes GR, Dixon RH, Silver D. Heparin induced thrombocytopenia with thrombotic and hemorrhagic manifestations. Surg Gynecol Obstet 136:409-416, 1973.

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HIT: Pathophysiology

HIT is a well-recognized complication of heparin therapy. Approximately 1 – 3% of patients exposed to heparin for greater than a week will experience a drop in their baseline platelet count. Typically, the decrease in patient platelet counts is identified between five to ten days following initiation of therapy. In early studies, it was suggested patients made antibodies to heparin. More recently, the antigenic target has been identified as a complex of platelet factor 4 and heparin.

Platelet Factor 4 (PF4) is found in the platelet dense bodies and is released to the ambient plasma upon platelet activation. Patients may produce IgG which reacts with Heparin-PF4. The complex of antibody-heparin-PF4 binds to FcIIa platelet receptors. Binding to receptors on endothelial cells will also occur. With cellular activation of endothelial cells and platelets, a significant prothrombotic state is induced. With activation of platelets and endothelial cells, “platelet/endothelial microparticles” are released into the circulating blood.

The generation of thrombin is an essential component in inducing thromboembolic events in the context of cellular activation. Thrombin generation may continue even after the discontinuation of heparin therapy. Consequently, other anticoagulants may be required in the management of these patients (e.g., Hirudin). Hirudin will inhibit thrombin bound to fibrin or fibrin degradation products.

Fratantoni and colleagues described a patient in 1975 who developed severe thrombocytopenia and complicating pulmonary emboli while being treated with unfractionated heparin for deep vein thrombosis. Laboratory studies utilizing patient sera found aggregation and serotonin release when patient sera was exposed to normal platelets in the presence of heparin. Subsequent studies by several groups confirmed the presence of heparin-induced platelet activating antibodies.

It was also observed that there was a temporal element characteristic of the delayed onset of HIT. In most cases, patients with HIT experienced thrombocytopenia after five or more days of heparin exposure. Platelet counts in heparin treated patients may vary widely. Occasionally, the platelet count drops below 50,000/uL. More frequently, the platelet counts are between 50 and 90,000/uL. It is also important to recognize patients who present with HIT may have platelet counts within the normal reference interval. HIT may be defined as a decrease of platelet count to less than 100,000 per uL or a decrease in baseline platelet count greater than 40%. An important caveat is to obtain a baseline platelet count prior to initiation of heparin therapy.

Table 3 summarizes the differential diagnosis for autoimmune thrombocytopenia.

Table 3: Autoimmune Thrombocytopenia*

• Idiopathic Thrombocytopenic Purpura (TTP)

• Autoimmune Thrombocytopenias:      Associated with Other Diseases– SLE and other connective tissue disorders – Antiphospholipid antibodies – Thyroid disorders (e.g., Hashimoto’s, Graves disease) – Lymphoproliferative disorders – Systemic infections (HIV, mononucleosis) – Sarcoidosis

• Drug-Induced Thrombocytopenia– Heparin – Colloidal gold – Quinidine/quinine – Sulfa antibiotics – Penicillin, ampicillin, cephalosporin

* Modified from Barbui T et al. Seminars in Clinical Immunology 2:33-43, 1998

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HIT: Type I and Type II

The benign form of HIT (Type I) is often seen early in the course of heparin therapy. Type I HIT is characterized by a transient mild decrease in platelet counts often immediately after initiation of heparin. Frequently, this form of HIT is not clinically detected. Type I HIT is due to a direct effect of heparin on platelets resulting in platelet aggregation and transient splenic sequestration. Thromboembolic events are not associated with Type I HIT.

HIT (Type II) is usually seen in the context of prolonged exposure (or re-exposure) to heparin leading to clinical thromboembolic complications. There are often marked drops in the baseline platelet count (less than 100,000 platelets per uL). HIT Type II is associated with a mortality of approximately 30% and patient morbidity (e.g., loss of limb, stroke, etc.) of 20%. In cases of patients who have recent heparin exposure, HIT may occur within hours following initiation of heparin therapy.

Table 4 compares and contrasts the differences between Type I and Type II HIT.

Table 4: Heparin-Induced Thrombocytopenia (HIT)
	TYPE I	TYPE II
Onset	Early (within 2 – 5 days)	Late* (after 6 days)
Platelet Count	Mild decrease; > 100,000/uL	Severe decrease; < 100,000/uL
IgG Antibody	No	Yes
Symptoms	Asymptomatic	Thrombosis
Duration	Transient	Persistent
Incidence	10 – 20%	< 1% – 30%
* Note Type II HIT may occur early in course of heparin administration if the patient has been previously exposed to heparin (e.g. recent PCTA followed by coronary artery bypass grafts). In patients with previous heparin exposure, HIT may occur within hours after initiation of therapy.

Variables that affect the frequency of Type II HIT are outlined in Table 5 and include route of administration. There is a greater incidence of HIT Type II with IV administration when compared to subcutaneous administration. Another variable is source of heparin, which can be of either bovine or porcine origin. Bovine heparin has an increased frequency of HIT when compared to porcine heparin. Additionally, while LMWH has a lower incidence of HIT Type II when compared to unfractionated heparins, it is important to appreciate that LMWH may be associated with HIT.

Table 5: HIT: Variables

• Source of heparin (bovine, porcine)• Unfractioned heparin• Low molecular wt. heparin (LMWH)• Duration of treatment• Route of administration• Diluent in IV tubing• History of past heparin exposure• Platelet count (e.g., transient HIT-Type 1)

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HIT: Clinical Presentation

HIT patients may present with thrombocytopenia or thromboembolic events during heparin administration. In the thrombocytopenic patient, it is necessary to exclude other causes of thrombocytopenia including: sepsis, dilutional effect, decreased bone marrow production and exposure to drugs which may cause drug-induced thrombocytopenia. As noted, Type I heparin-induced thrombocytopenia is not associated with clinical thromboembolic events. (Table 4)

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Evidence Supporting Antibody Mediated Process

In today’s hospital environment, heparin exposure is very common (keep open lines, dialysis patients, patients receiving subcutaneous LMWH, use of LMWH in outpatient management of DVT, etc.). (Table 5)

Another scenario which may be catastrophic involves patients presenting with chest pain. Following catheterization of coronary arteries with either stent placement or balloon procedures, patients are often sent home after a relatively brief period of time in the hospital. Not infrequently, patients may return complaining of chest pain several weeks after the initial invasive procedures. If these patients are re-exposed to heparin, they may develop antibodies to heparin/PF4 complex. The anamnestic response of H-PF4 antibodies may lead to rapid onset of thrombosis and, in some cases, fatalities.

As part of the pathophysiology in this setting, the platelet FcRIIa receptor is necessary for platelet aggregation response. As noted above, IgG binds to platelet factor 4 complexed with heparin. Other plasma proteins may also serve as antigenic targets in the context of HIT. These proteins include: interleukin-8, neutrophil activating peptide-2 (NAP-2), ß2 thromboglobulin, and PF4. (Table 6)

HIT antibodies also react with heparin sulfate PF4 complexes on the surface of endothelial cells. As a consequence, there is an up-regulation of endothelial cell tissue factor and release of endothelial cell microparticles into the circulation.

Table 6: HIT: Protein-Polysaccharide Complex(es)
Polysaccharides	Proteins
Heparin (UFH)	PF4
LMWH	IL8
Pentosan Polysulfate	NAP-2
Chondrotin Sulfate	BTG
Polysulfated Oligosacch
Platelet Factor 4 (PF4) Interleukin-8 (IL8) Neutrophil Activating Peptide-2 (NAP-2) Beta2 Thromboglobulin (BTG)

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HIT: Laboratory Diagnosis

Aggregation Studies
A variety of assays have been described to verify the presence of HIT in patients with appropriate clinical findings/settings. For many years, platelet aggregation studies have been the most frequently performed test to identify HIT patients. The aggregation studies are cumbersome, requiring [control A] patient poor plasma (PPP); normal donor platelet rich plasma [control B]; donor platelet poor PPP, normal donor PRP + heparin (0.5 units per mL) to rule out nonspecific effect of heparin on normal donor PRP.

The patient’s platelet poor plasma plus normal donor platelet rich plasma and heparin at two concentrations, 0.5 units per mL and 100 units per mL, is the standard procedure for platelet aggregation studies. A positive test requires greater than 20% aggregation when compared to the normal control. Aggregation between 10 – 20% is interpreted as an equivocal result. The aggregation response is often delayed so it is important to monitor aggregation for at least 15 minutes.

REMEMBER, A NEGATIVE TEST DOES NOT RULE OUT HIT!!
When the clinical picture is strongly suggestive of HIT and the aggregation studies are negative, a repeat series of aggregation tests three to seven days later may yield a positive result.

Serotonin Release Assay

The serotonin release assay is the “gold standard” for identifying HIT. Unfortunately, this procedure is time-consuming and requires the use of radioisotopes. The serotonin release assay is performed in only a few laboratories and, because of the assay requirements, it is quite expensive.

Table 7: HIT Testing
HIT ELISA • PF4 + Heparin Coated Plate • Anti-IgG, IgA, IgM • Sensitivity: 80 – 90% • Specificity       – Pos. Pred. Value: 50 – 70%       – Neg. Pred. Value: 80 – 90%
HIT Testing: Comparative Analysis	Pred. Values
	Sensitivity	Specificity	Pos.	Neg.
PF4-H ELISA	97%	86%	93%	91%
Serotonin Release	88%	100%	100%	81%
Plt. Aggreg.	91%	77%	89%	81%

ELISA Assay

Other assays have been developed using ELISA techniques to detect HIT antibodies. Jean Amiral was the first to describe an ELISA assay for HIT diagnosis. (Table 7) Commercially available ELISA assays for antibodies to PF-4-heparin are available. Warde presently utilizes GTI-PF4 ELISA as the only on site assay for HIT. Microtiter plates are coated with a mixture of PF4 and heparin. Patient plasma is added to the microtiter plate wells and the binding of antibodies to Heparin-PF4 is identified by anti-human IgG.

The rate of false positives in ELISA assays, including GTI, is high. As many as 15% of patients following cardiotomy develop these antibodies. Most of these patients do not have clinical evidence of HIT and will not develop HIT if re-exposed to heparin.

A positive result in the absence of a compatible clinical setting or as a screening test has no value. However, the assay is highly sensitive (about 90%) for HIT. Therefore the test should only be employed with compatible clinical findings (at least 30% drop in platelet count, thrombosis, and/or the absence of another etiology for these findings, such as immune or drug induced thrombocytopenia, sepsis, or major surgery). In a compatible clinical setting, greater than one week following heparin exposure, a positive GTI-PF4 ELISA result supports an impression of HIT and warrants the management steps discussed in the next section. Under those same conditions, a negative result argues that other causes for the clinical findings be investigated as, or before, the inconvenient, potentially toxic, and expensive therapeutic measures for HIT be undertaken. This test is not 100% sensitive nor is it available “STAT”. Management of the patient frequently must occur before results are known.

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HIT: Management

The most important aspect of HIT management is discontinuing all exposure to Heparin (e.g., heparin line flushes, prophylactic and therapeutic heparin, heparin-coated catheters).

A variety of alternative anticoagulants are now available including recombinant Hirudin (Rifluden®), Danaparoid (Orgaran®), Argatroban (Novastan®), and Iib/IIIa inhibitors. The use of Coumadin in the management of the patient with HIT has a potential for inducing venous limb gangrene syndrome. Consequently, alternative anticoagulants are indicated as the initial approach to anticoagulating the HIT patient. Once there is significant resolution of HIT, coumadin may be used for long-term control of the prothrombotic state.

There are a number of innovative drugs being developed by various pharmaceutical companies. Recently, an oral anti-Xa drug has been approved by the FDA. This drug is a synthetic pentasaccharide (Org 31540/SR90107A)* Preliminary studies suggest this drug may improve the risk benefit ratio for prophylaxis to prevent deep vein thrombosis.

* ARIXTRA® (fondaparinux sodium) injection

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